Differential activation of tumor necrosis factor receptors distinguishes between brains from Alzheimer's disease and non-demented patients

Abstract

We reported that tumor necrosis factor receptor I (TNFRI) is required for neuronal death induced by amyloid-beta protein in the Alzheimer's disease (AD) brain. However, whether TNF receptor subtypes are expressed and activated differentially in AD brains compared to non-demented brains remains unclear. Our studies on Western blot and ELISA measurements demonstrated that TNFRI levels are increased whereas TNFRII levels are decreased in AD brains compared to non-demented brains (p <0.05). Immunohistochemical results demonstrated that both TNFRI and TNFRII are expressed in neurons in AD and non-demented brains. However, in situ hybridization studies showed little change in the mRNA levels of either type of TNF receptor in the neurons of AD brains compared to non-demented brains. To examine whether different levels of TNF receptors in AD brains are correlated with the alteration of functional binding of TNF receptors, by using 125I-TNF-alpha binding technique, we found that, in AD brains, 125I-TNF-alpha binding affinity to TNFRI is increased, whereas binding affinity to TNFRII is decreased (p < 0.01). These studies reveal a novel observation of abnormal TNF receptor activation in AD brains. Differential TNF receptor protein levels and binding affinities suggest distinct pathogenic mechanisms of neurodegeneration in the AD brain.

Fig. 1

Different levels of TNFRI and TNFRII protein in AD brains. The frontal cortex region in AD and ND individuals were homogenated, and TNFRI and TNFRII protein levels were measured specifically by Western blot (A and B) and ELISA (C). Western blot studies on TNFRI and II, synaptophysin, and neuron specific enolase (NSE) in AD and ND brains are shown in (A). (B) Data represent the mean (± SD) quantification analysis using densitometry imaging (FluorChem 8900) showing that TNFRI immunoreactivity was increased while TNFRII was decreased in AD brains compared to ND, whereas synaptophysin was decreased about 24%. There is no significant change in NSE protein levels. β-actin was used as a loading control. (C) ELISA assay showed a 43% reduction in TNFRII and a 28% increase in TNFRI in AD frontal tissue compared to that found in ND. Data in bar graphs represent mean±SEM. All results were repeated at least three times from independent experiments. *p < 0.05; **p < 0.01.

Fig. 2

Neuronal expression of TNFRI and TNFRII mRNA in human brains using in situ hybridization. AD frontal cortex sections were hybridized to ³⁵S-labeled sense and antisense cRNA probes for TNFRI and TNFRII. Resultant autoradiographs were used directly as negatives for photographic enlargement of frontal cortex. All of pictures in the left panel (A,C,E,G) are hybridization by antisense cRNA probes whereas the pictures in the right panel (B,D,F,H) are sense hybridization as controls. The frontal cortex sections from a 79-year-old male with AD hybridized with antisense cRNA probes for TNFRI (A) and TNFRII (C). Note the specific hybridization over the cortical strip of the frontal cortex. Furthermore, (A) and (C) also demonstrate that specific hybridization was noted in pyramidal neurons and interneurons in this area. Similarly, the frontal cortex sections from a non-demented aged matched control brain (82-year-old male) hybridized with antisense RNA probes for TNFRI (E) and TNFRII (G). We found similar neuronal distribution of both TNFRI and II transcripts, hybridized specifically in pyramidal neurons and interneurons in this brain region.

Fig. 3

TNFRI and TNFRII protein expressions in AD and ND brains using immunohistochemistry. (A) TNFRI protein expression is increased in AD cortical tissues and much higher expression of TNFRI was observed on the dendrites. Compared to ND brains, there is an increased TNFR1 level in AD cortical tissues along with a decreased number of neurons (NeuN staining). There is little TNFR1 expression in activated astrocytes in ND and AD brains, even in the over-activated astrocytes in AD patients. Bar stands for 20 μm. (B) The TNFRII protein expression is decreased in AD cortical tissues compared to ND controls. A portion of neurons in the cortex are showed with strong TNFRII-positive immunostaining on the cell membrane around neuronal nuclei (verified by NeuN staining). Astrocytes have little TNFRII expression with immunostaining in both ND and AD brains. Bar stands for 20 μm.

Fig. 4

Scatchard analysis of ¹²⁵I-TNF-α binding in human brain tissue from AD and ND patients (A) and representative competition curve for TNF-α inhibition of 100 pM ¹²⁵I-TNF-α binding in human brain tissue from AD and ND patients (B). Incubation conditions were described in Methods. Computer analysis of the binding data revealed that competition curve is best described by two-components binding model, with different sets of IC₅₀ values.