Serotonin 2A Receptor Autoantibodies Increase in Adult Traumatic Brain Injury In Association with Neurodegeneration

Abstract

Objective: Traumatic brain injury (TBI) is associated with an increased risk of late neurodegenerative complications via unknown mechanisms. Circulating neurotoxic 5-hydroxytryptamine 2A receptor (5-HT2AR) autoantibodies were reported to increase in subsets of obese type 2 diabetes having microvascular complications. We tested whether 5-HT2AR autoantibodies increase in adults following traumatic brain injury in association with neurodegenerative complications.

Methods: Plasma from thirty-five middle-aged and older adult veterans (mean 65 years old) who had suffered traumatic brain injury was subjected to protein-A affinity chromatography. The resulting immunoglobulin (Ig) G fraction was tested for neurotoxicity (acute neurite retraction, and accelerated cell death) in mouse N2A neuroblastoma cells or for binding to a linear synthetic peptide corresponding to the second extracellular loop region of the human 5-HT2A receptor.

Results: Nearly two-thirds of traumatic brain injured-patients harbored 5-HT2AR autoantibodies in their circulation. Active TBI autoantibodies caused neurite retraction in mouse N2A neuroblastoma cells and accelerated N2A cell loss which was substantially prevented by co-incubation with a two hundred and fifty nanomolar concentration of M100907, a highly selective 5-HT2AR antagonist. Antagonists of RhoA/Rho kinase and Gq11/phospholipase C/inositol triphosphate receptor signaling pathways blocked TBI autoantibody-induced neurite retraction. Following traumatic brain injury, autoantibody binding to a 5-HT2A receptor peptide was significantly increased in patients having co-morbid Parkinson's disease (n=3), dementia (n=5), and painful neuropathy (n=8) compared to TBI subsets without neurologic or microvascular complication (n=20). Autoantibody titer was significantly elevated in TBI subsets experiencing multiple neurotraumatic exposures vs. single TBI. Plasma white blood cell, a marker of systemic inflammation, correlated significantly (correlation coefficient r =0.52; P < 0.01) with, 5-HT2A receptor peptide binding of the TBI-autoantibody.

Conclusion: These data suggest that circulating neurotoxic 5-hydroxytryptamine 2A receptor agonist autoantibodies increase in adults following traumatic brain injury in association with late neurodegenerative complications.

Figure 1:

Enzyme-linked immunoassay using 5-HT2A receptor second extracellular loop region linear synthetic 18-mer; mean binding (indicated by horizontal lines) was significantly increased in autoantibodies (1/40th dilution) from TBI patients having co-morbid painful neuropathy (PN), Parkinson’s disease (PD), dementia and diabetic retinopathy (Retnpy) compared to twenty TBi patients who lacked neurologic, diabetic microvascular complication (TBI uncompl). One non-diabetic TBI patient with highest binding had both Parkinson’s disease and dementia. CVA-cerebrovascular accident, Retnpy-diabetic retinopathy, Neph(ropathy), MDD-major depressive disorder; Total N exceeds thirty-five because many of the same patients had more than one co-morbidity, e.g. dementia and retinopathy, and painful neuropathy and nephropathy.

Figure 2:

Correlation between white blood cell count and 5-HT2A receptor peptide binding in the protein A eluates from A) 35 patients with TBI or B) 47 patients with type 2 diabetes and no TBI

Figure 3:

Protein-A eluate fraction in representative TBI patient was incubated with N2A cells for 24 hours at indicated dilutions. Cell survival was determined with MTT assay as described in Materials and Methods B)A one-fiftieth dilution of the protein-A eluate fraction from eight different TBI plasmas was incubated with N2a cells in the presence or absence of the highly selective 5-HT2AR receptor antagonist M100907 (250 nM) or the alpha-1 adrenergic receptor antagonist prazosin (850 nM)(n=5 patients). Cell survival (after 24 hours) was determined using MTT assay. * P< 0.01 compared to TBI autoantibodies alone.

Figure 4:

Multiple TBI exposure is associated with increased autoantibody titer and 5-HT2AR peptide binding potency compared to single uncomplicated TBI;* P< 0.01; Aab-autoantibody; B) Protein-A eluate in type 2 diabetic dementia patient having multiple microvascular complications bind with high potency and titer to both 5-HT2AR peptide and to purified neuronal-cell derived heparan sulfate proteoglycan (HSPG).

Figure 5:

Correlation between 5-HT2AR peptide and neuronal HSPG binding in the protein-A eluates from thirty-one of thirty-five TBI patients.