Functional autoantibodies in patients with different forms of dementia

Abstract

Dementia in general and Alzheimer's disease in particular is increasingly seen in association with autoimmunity being causatively or supportively involved in the pathogenesis. Besides classic autoantibodies (AABs) present in dementia patients, there is the new autoantibody class called functional autoantibodies, which is directed against G-protein coupled receptors (GPCRs; GPCR-AABs) and are seen as pathogenic players. However, less is known about dementia patients' burden with functional autoantibodies. We present here for the first time a study analyzing the prevalence of GPCR-AABs in patients with different dementia forms such as unclassified, Lewy body, vascular and Alzheimer's dementia. We identified the GPCR-AABs' specific targets on the receptors and introduced a neutralization strategy for GPCR-AABs. Patients with Alzheimer’s and vascular dementia carried GPCR-AABs targeting the second loop of the alpha1- and the first loop of the beta2-adrenergic receptors (α1-AABs; β2-AABs). The majority of patients with Lewy body dementia lacked any of the GPCR-AABs. In vitro, the function of the dementia-associated GPCR-AABs could be neutralized by the aptamer BC007. Due to the presence of GPCR-AABs in dementia patients mainly in those suffering from Alzheimer's and vascular dementia, the orchestra of immune players in these dementia forms, so far preferentially represented by the classic autoantibodies, should be supplemented by functional autoantibodies. As dementia-associated functional autoantibodies could be neutralized by the aptamer BC007, the first step was taken for a new in vivo treatment option in dementia patients who were positive for GPCR-AABs.

Fig 1. Chart of the bioassay using spontaneously beating cultured neonatal rat cardiomyocytes for the characterization of autoantibodies directed against G-protein-coupled receptors (GPCR-AABs).

Fig 2. Measurement strategy for autoantibodies directed against G-protein coupled receptors (GPCR-AABs) in patients’ IgG using the bioassay of cultured spontaneously beating neonatal rat cardiomyocytes.

The IgGs’ chronotropic activity resulting from the presence of positive and negative chronotropic GPCR-AABs was monitored. For GPCR-AAB differentiation and activity calculation, the bioassay was performed after the successive addition of blockers against the AT1 (0.1 μmol/l Losartan), β2-adrenergic (0.1 μmol/l ICI 118551), α1-adrenergic (0.1 μmol/l Prazosin) and endothelin A receptors (0.1 μmol/l BQ123) and (for independent confirmation) after IgG pre-treatment separately with the respective blockers.

Fig 3. Activity of autoantibodies directed against the α1-adrenergic (blue), β2-adrenergic (red) and endothelin A receptor (green) in the serum of patients with unclassified, Lewy body, vascular and Alzheimer’s dementia.

Box plots are plotted indicating median and interquartile range (IQR; 25th and 75th percentiles); whiskers have ends that represent the largest and smallest values inside 1.5 times the IQR, alongside outliers (open circles) that are values placed between 1.5 and 3 times the IQR, and extremes (stars) placed more than 3 times the IQR. Lower limit of detection (LLD) for positive and negative chronotropic activity = 4.0 U and -4.0 U; cut off for GPCR-AAB positivity = 8.0 U for positive and -8.0 U for negative chronotropic GPCR-AAB activity.

Fig 4. Autoantibodies directed against the endothelin A receptor (ETA-AABs) of patients with vascular dementia target the second extracellular receptor loop.

Using the bioassay of spontaneously beating cultured neonatal rat cardiomyocytes, the chronotropic activity of the patients’ IgG (n = 3), either untreated or pre-incubated with peptides representing the first (¹³⁴LPINVFKLLAGRWPFDHNDFGVFLCKL¹⁶⁰), second (²²⁹FEYRGEQHKTCMLNATSKFMEFYQDVKD²⁵⁶), and third extracellular receptor loop (³²⁹KKTVYNEMDKNRCELLLSFLL³⁴⁸), was measured. Values below the low limit of detection (LLD) were displayed as half range values. LLD = -4 beats/min; cut-off (separating healthy from disease subjects) = − 8 beats per/min.

Fig 5. Mapping of the second extracellular loop of the endothelin A receptor for epitope localization targeted by autoantibodies directed against the endothelin A receptor (ETA-AABs) of patients with vascular dementia.

Using the bioassay of spontaneously beating cultured neonatal rat cardiomyocytes, the ETA-AAB activity of the patients’ IgG (n = 3) untreated or pre-incubated with peptides that overlapped to represent the second extracellular endothelin A receptor loop (P1: FEYRGEQ, P2: EQHKTCM, P3: MLNATSK, P4: SKFMEFY, P5: FYQDVKD) was measured. Values below the low limit of detection (LLD) were displayed as half range values. LLD = − 4 beats/min; cut-off (separating healthy from disease subjects) = − 8 beats per/min.

Fig 6. A and B. Influence of the aptamer BC 007 on the activity of autoantibodies directed against the G-protein coupled receptors (GPCR-AABs) specifically those to the β2-adrenergic (β2-AABs), α1-adrenergic (a1-AABs) and endothelin A receptor (ETA-AABs) in patients with (A) vascular (β2-AABs, α1-AABs, ETA-AABs) and (B) Alzheimer’s dementia (β2-AABs, α1-AABs).

The total chronotropic activity of the patients’ IgG as well as the activities related to each autoantibody on spontaneously beating cultured neonatal rat cardiomyocytes isolated from the serum of all 4 patients in the absence (colored columns) and presence (0.1 μM) of BC 007 (grey columns) are demonstrated. For each one of the patients with vascular and Alzheimer’s dementia, the GPCR-AAB activity in the presence of a scrambled 15 mer aptamer is additionally demonstrated. Values below the low limit of detection (black line = LLD) were displayed as half range values. LLD = 4 beats/min and—4 beats/min, respective.