Circulating Neurotoxic 5-HT2A Receptor Agonist Autoantibodies in Adult Type 2 Diabetes with Parkinson's Disease

Abstract

Aims: To test whether circulating neurotoxic autoantibodies increase in adult type 2 diabetes mellitus with Parkinson's disease (PD) or dementia. To identify the G-protein coupled receptor on neuroblastoma cells mediating neural inhibitory effects in diabetic Parkinson's disease plasma autoantibodies. To determine the mechanism of accelerated neuroblastoma cell death and acute neurite retraction induced by diabetic Parkinson's disease and dementia autoantibodies.

Methods: Protein-A eluates from plasma of twelve older adult male diabetic patients having Parkinson's disease (n=10) or dementia (n=2), and eight age-matched control diabetic patients were tested for ability to cause accelerated N2A neuroblastoma cell death and acute neurite retraction. Specific antagonists of G protein coupled receptors belonging to the G alpha q subfamily of heterotrimetric G-proteins, the phospholipase C/inositol triphosphate/Ca2+ pathway, or the RhoA/Rho kinase pathway were tested for ability to block diabetic Parkinson's disease/dementia autoantibody-induced neurite retraction or N2A accelerated cell loss. Sequential Liposorber LA-15 dextran sulfate cellulose/protein-A affinity chromatography was used to obtain highly-purified fractions of diabetic Parkinson's disease autoantibodies.

Results: Mean accelerated neuroblastoma cell loss induced by diabetic Parkinson's disease or dementia autoantibodies significantly exceeded (P = 0.001) the level of N2A cell loss induced by an identical concentration of the diabetic autoantibodies in control patients without these two co-morbid neurodegenerative disorders. Co-incubation of diabetic Parkinson's disease and dementia autoantibodies with two-hundred nanomolar concentrations of M100907, a highly selective 5-HT2AR antagonist, completely prevented autoantibody-induced accelerated N2A cell loss and neurite retraction. A higher concentration (500 nM-10μM) of alpha-1 adrenergic, angiotensin II type 1, or endothelin A receptor antagonists did not substantially inhibit autoantibody-induced neuroblastoma cell death or prevent neurite retraction. Antagonists of the inositol triphosphate receptor (2-APB, 50μM), the intracellular calcium chelator (BAPTA-AM, 30 μM) and Y27632 (10 μM), a selective RhoA/Rho kinase inhibitor, each completely blocked acute neurite retraction induced by sixty nanomolar concentrations of diabetic Parkinson's disease autoantibodies. Co-incubation with 2-APB (1-2 μM) for 8 hours' prevented autoantibody-induced N2A cell loss. The highly-purified fraction obtained after Liposorber LA/protein-A affinity chromatography in hypertriglyceridemic diabetic dementia and Parkinson's disease plasmas had apparent MWs > 30 kD, and displayed enhanced N2A toxicity requiring substantially higher concentrations of 5-HT2AR antagonists (M100907, ketanserin, spiperone) to effectively neutralize.

Conclusion: These data suggest increased autoantibodies in older adult diabetes with Parkinson's disease or dementia cause accelerated neuron loss via the 5-hydroxytryptamine 2 receptor coupled to inositol triphosphate receptor-mediated cytosolic Ca2+ release.

Keywords: 5-HT2A receptor; Parkinson’s disease; autoantibodies; dementia; diabetes.

Figure 1

Diabetes Parkinson’s disease (n=10) or dementia (n=2) auto antibodies (60 nM) caused significant N2a neuroblastoma cell loss after 24 hrs incubation compared to identical concentration of autoantibodies in the protein-A eluate of plasma in diabetic patients without neurodegenerative disorder (control EL). B) Neuroblastoma cell loss induced by (60 nM) concentration of diabetic PD (n=5) or dementia (n=1) auto antibodies was completely prevented by co incubation with 200 nM concentration of M100907, a highly selective 5-HT2A receptor antagonist. N2A cells were incubated for 24 hours at 37 degrees. Cell number was determined as described in Methods. Results are mean ± SEM.

Figure 2

Diabetic Parkinson’s disease (n=2) or dementia autoantibodies (n=1) caused significant suppression of N2A cell neurite extension compared to control cells (A); Mean N2A neurite length suppression induced by three different diabetic PD autoantibodies was nearly completed prevented by co-incubation with 200 nM M100907, a highly selective 5-HT2A receptor antagonist (B). Potent, time-dependent suppression of N2A neurite outgrowth by diabetic dementia (Pt 1) autoantibodies was partially prevented by co-incubation with M100907(C).

Figure 3

Neuroblastoma cell toxicity was present in the purified fraction eluting from protein A following Liposorber/protein-A sequential chromatography, but was lacking in the other fractions (A). The highly purified protein-A eluate fraction of diabetic dementia plasma caused significantly greater dose-dependent suppression of N2A neurite outgrowth compared to the Liposorber eluate fraction from the same patient (B). Dose-dependent N2A cell toxicity in the protein-A eluates from diabetic dementia (Pt 1) and PDD (Pt 3) significantly exceeded toxicity in the corresponding Liposorber DMT eluate fraction (C). ** P < 0.01; * P < 0.05.

Figure 4

The neuroblastoma cell toxicity in the highly-purified Liposorber/protein-A eluates of diabetic dementia and Parkinson’s disease with dementia was significantly blocked by co-incubation with 500 nM concentrations of A) M100907, B) spiperone, or C) ketanserin.

Figure 5

Peak neurotoxicity in the highly purified diabetic dementia autoantibodies was retained following dialysis on a 30 kD MW cutoff membrane (A). The N2A neurotoxicity in PD and diabetic dementia auto antibodies was completed prevented by co-incubation with 1–2 μM concentrations of the IP3R blocker 2-APB(B). Mean N2A neurotoxicity in autoantibodies from five different diabetic PD or dementia patients was not significantly affected by co-incubation with Losartan, bosentan or prazosin (C).

Figure 6

Heating (65 deg C x 30 mins) caused gain in N2A neurotoxicity in a subset of six diabetic patients with co-morbid PD (n=5) or dementia (n=1) (A). There was no effect of heating on N2A toxicity in protein-A eluates from two diabetic control patients without neurodegenerative disease co morbidity (B). Heating caused a modest gain in N2A proliferation (n=3) or a loss in N2A toxicity in a second subset of six diabetic patients with co-morbid PD (n=5) or dementia (n=1) (C).

Figure 7

Diabetic PD autoantibodies (60 nM) caused contraction in HL-1 atrial cardiomyocytes within 5 minutes of application. Photomicrographs (100 x magnification) were captured with a Nikon TMS microscope. A) Control HL-1 cells prior to addition of diabetic PD autoantibodies; B) corresponding images of the same HL-1 cells 5 minutes after the application of diabetic PD (Pt 4) autoantibodies. Higher magnification of cells (A) shown in inset by rectangle (C, D), diamond (E, F) or arrow (G, H) before (C, E, G) and five minutes after (D, F, H) the addition of PD autoantibodies. Similar results were obtained in experiments with two different diabetic PD patients autoantibodies.