Functional autoantibodies in patients with different forms of dementia

<div><p>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 first loop of the alpha1- and the second loop of the beta2-adrenergic receptors (α1-AABs; β2-AABs). Nearly all vascular dementia patients also carry autoantibodies targeting the endothelin A receptor (ETA-AABs). The majority of patients with Lewy body dementia lacked any of the GPCR-AABs. <i>In vitro</i>, 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 <i>in vivo</i> treatment option in dementia patients who were positive for GPCR-AABs.</p></div

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

<p>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 (</p><p>¹³⁴LPINVFKLLAGRWPFDHNDFGVFLCKL¹⁶⁰</p>), second (<p>²²⁹FEYRGEQHKTCMLNATSKFMEFYQDVKD²⁵⁶</p>), and third extracellular receptor loop (<p>³²⁹KKTVYNEMDKNRCELLLSFLL³⁴⁸</p>), 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.<p></p

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.

<p>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.</p

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.

<p>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.</p

Dementia-associated autoantibodies directed against G-protein coupled receptors (GPCR-AABs) such as those directed against the α1-adrenergic (α1-AABs), β2-adrenergic (β2-AABs) and endothelin A receptor (ETA-AABs) related to their target (extracellular receptor loop) with the specific epitope.

<p>Dementia-associated autoantibodies directed against G-protein coupled receptors (GPCR-AABs) such as those directed against the α1-adrenergic (α1-AABs), β2-adrenergic (β2-AABs) and endothelin A receptor (ETA-AABs) related to their target (extracellular receptor loop) with the specific epitope.</p

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).

<p>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.</p

The pattern of functional autoantibodies in patients with unclassified, Lewy body, vascular and Alzheimer’s dementia.

<p>Serum positivity and negativity are demonstrated for autoantibodies directed against α1-adrenergic (α1-AABs), β2-adrenergic (β2-AABs) and endothelin A receptors (ETA-AABs); a score was calculated for the integrated activity assessment of the three autoantibodies directed against G-protein coupled receptors (GPCR-AABs). For the score calculation, the GPCR-AAB activities were summarized based on: 0 point = GPCR-AAB level LLD < cut-off; 2 points = GPCR-AAB level > cut-off.</p

ADRB1-AB epitope mapping experiments.

<p><b>A:</b> The change in beats per minute in rat cardiomyocytes upon delivery of 0.335 nM ADRB1-AB and the influence of various mapping peptides on the ADRB1-AB caused beat rate increase. All peptides (300 nM final concentration) were pre-incubated with the ADRB1-AB sample for 1 hour before adding to the cell culture flask and data is represented as mean ± SD of 6 technical replicates. <b>B:</b> An ELISA using the immobilized immunogen peptide (0.1 μg/well) for binding ADRB1-AB (5 nM), was performed with mixtures of ADRB1-AB and the corresponding mapping peptide (concentration as indicated in the figure). The mixtures were diluted in PBS and incubated for 1 h before adding onto cardiomyocyte cell culture medium preloaded ELISA wells. Anti-goat IgG-POD and H₂O₂/TMB served for detection and quantification. The presented data show the absolute measured extinction values free of any computation of two technical replicates.</p

Fig 4 -

Influence of the Aβ peptides on the clenbuterol induced β2-adrenoceptor desensitization: The desensitization of the β-AR response by clenbuterol, was not influenced by the short chain Aβ peptides (a). However, the long chain Aβpeptides Aβ 10–37, Aβ1–40, and Aβ1–42 prevent the desensitization and exert a permanent stimulation of the β2-AR signal cascade (b).</p

Direct ELISA for ADRB1-AB detection.

<p>Quantification of free soluble ADRB1-AB of varying concentration as indicated in the abscissa. The presented data show the mean of the absolute measured extinction values free of any computation. <b>A:</b> ADRB1-AB spiked into human control serum (“100% human serum”), of two independent experiments, one in triplicate and one in duplicate, using different ADRB1-AB batches. The inset shows the data in logarithmic display. <b>B and D:</b> ADRB1-AB spiked in 50% human serum and <b>C:</b> ADRB1-AB spiked in 50% goat serum (species identical matrix material). Here the presented data are mean of two independent experiments. <b>E:</b> Varying concentrations of goat serum of 1%, 5%, 10%, 20%, 30%, 40% and 50% were spiked with a constant concentration of 25 nM ADRB1-AB. The presented data are the mean of one experiment with two technical replications. Anti-goat IgG-POD (1:10,000)/TMB served for detection and quantification. Immunogen-peptide uncoated wells served as a control.</p