Neurogranin binds α-synuclein in the human superior temporal cortex and interaction is decreased in Parkinson's disease

Abstract

Neurogranin is a calmodulin binding protein that has been implicated in learning and memory, long-term potentiation and synaptic plasticity. Neurons expressing neurogranin in the cortex degenerate in late stages of Parkinson's disease with widespread α-synuclein pathology. While analyzing neurogranin gene expression levels through rtPCR in brains of mouse models overexpressing human α-synuclein, we found levels were elevated 2.5 times when compared to nontransgenic animals. Immunohistochemistry in the cortex revealed colocalization between α-synuclein and neurogranin in mouse transgenics when compared to control mice. Coimmunoprecipitation studies in the superior temporal cortex in humans confirmed interaction between α-synuclein and neurogranin, and decreased interaction between α-synuclein and neurogranin was noticed in patients diagnosed with Parkinson's disease when compared to normal control brains. Additionally, phosphorylated neurogranin levels were also decreased in the human superior temporal cortex in patients diagnosed with Parkinson's disease and patients diagnosed with dementia with Lewy bodies. Here, we show for the first time that neurogranin binds to α-synuclein in the human cortex, and this interaction decreases in Parkinson's disease along with the phosphorylation of neurogranin, a molecular process thought to be involved in learning and memory.

Keywords: Calmodulin; Dementia with Lewy bodies; LTP; Neurogranin; Parkinson’s disease; α-Synuclein.

Fig. 1

Neurogranin expression levels. Neurongranin RNA levels in the brains of Tg mice over expressing human α-syn. Expression increased 2.5 times in Tg mice when compared to normal controls (Fig. 2; 2.58±1.21 vs. 0.98±0.45 relative expression, respectively; p=0.008; 2-sided t-test).

Fig. 2

Neurogranin interaction with α-synuclein in Tg mouse brain. Immunohistochemical neurogranin expression in nonTg mouse brains (green, (A)) when compared with α-syn (red, (B)) revealed no colocolization among cell bodies ((C) and (D)) but possible colocalization in the synapse. In Tg mouse brains, neurogranin (E) and α-syn (F) showed obvious yellow colocalization in cell bodies and synapes ((G) and (H)). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

Neurogranin and α-synuclein Western blot in superior temporal cortex in human brains. α-Syn protein levels as analyzed by Western blot and compared to actin were increased between PD and DLB brains when compared to normal controls (Fig. 4A and B) (^*=<.05; ^**=<.01). Neurogranin levels remained the same between normal controls, PD and DLB brains (Fig. 4A and C).

Fig. 4

Neurogranin interaction with α-synuclein in human superior temporal cortex. In human superior temporal cortex, when immunoprecipitated for neurogranin followed by Western blot of precepitate for α-syn, an interaction was observed in normal control, PD and DLB patients; additionally, reverse coIP, immunoprecipitation for α-syn and Western for neurogranin revealed interaction as well (A) (N=no antibody included in immunoprecipitation with DLB sample, B=blank, no sample included in immunoprecipitation). In (B) when immunoprecipitating with non specific IgG, no interaction was revealed, indicating that the interaction was specific for neurogranin and α-syn. In (C) pixel density analysis indicates a statistical reduction in PD brains in the interaction between neurogranin and α-syn (^**=p<.01). In (D) when comparing the levels of neurogranin from (C) with the levels of neurogranin in the coimmunoprecipitation assay as percent of normal control, normal neurogranin levels were slightly diminished in PD brains, whereas α-syn bound neurogranin levels were dramatically reduced by half in PD brains indicating a pathological difference in neurogranin-α-syn in this disease state. No difference was seen in DLB brains in normal neurogranin or neurogranin bound with α-syn.

Fig. 5

Phosphorylated neurogranin Western blot in the membrane and cytosolic fraction in superior temporal cortex in human brains. p-Ng protein levels as analyzed by Western blot and compared to actin were decreased in the membrane faction of superior temporal cortex in PD and DLB brains when compared to normal controls ((A) and (B)) (^*=<.05; ^**=<.01). p-Ng levels in the cytosolic fraction remained the same between normal controls, PD and DLB brains ((A) and (C)).