Anti-DPPX encephalitis: pathogenic effects of antibodies on gut and brain neurons

Abstract

Objective: To characterize pathogenic effects of antibodies to dipeptidyl-peptidase-like protein 6 (DPPX), a subunit of Kv4.2 potassium channels, on gut and brain neurons.

Methods: We identified a new patient with anti-DPPX encephalitis and analyzed the effects of the patient's serum and purified immunoglobulin G (IgG), and of serum of a previous patient with anti-DPPX encephalitis, on the activity of enteric neurons by voltage-sensitive dye imaging in guinea pig myenteric and human submucous plexus preparations. We studied the subcellular localization of DPPX by immunocytochemistry in cultured murine hippocampal neurons using sera of 4 patients with anti-DPPX encephalitis. We investigated the influence of anti-DPPX-containing serum and purified IgG on neuronal surface expression of DPPX and Kv4.2 by immunoblots of purified murine hippocampal neuron membranes.

Results: The new patient with anti-DPPX encephalitis presented with a 2-month episode of diarrhea, which was followed by tremor, disorientation, and mild memory impairment. Anti-DPPX-IgG-containing sera and purified IgG increased the excitability and action potential frequency of guinea pig and human enteric nervous system neurons. Patient sera revealed a somatodendritic and perisynaptic neuronal surface staining that colocalized with the signal of commercial anti-DPPX and Kv4.2 antibodies. Incubation of hippocampal neurons with patient serum and purified IgG resulted in a decreased expression of DPPX and Kv4.2 in neuronal membranes.

Conclusions: Hyperexcitability of enteric nervous system neurons and downregulation of DPPX and Kv4.2 from hippocampal neuron membranes mirror the clinical phenotype of patients with anti-DPPX encephalitis and support a pathogenic role of anti-DPPX antibodies in anti-DPPX encephalitis.

Figure 1. Clinical and paraclinical findings of a novel patient with anti–dipeptidyl-peptidase-like protein 6 encephalitis

(A) Time course of the disease, intensity of neurologic symptoms, Montreal Cognitive Assessment (MoCA) test scores, anti–dipeptidyl-peptidase-like protein 6 (DPPX) antibody titers, and treatments of a newly identified patient (patient 1) with anti-DPPX encephalitis. (B) T1-weighted axial MRI demonstrates no significant atrophy (upper left). PET with F-18-fluorodeoxyglucose (FDG-PET) as marker of synaptic activity shows strongly reduced activity bilaterally in the heads of the caudate nuclei and mild to moderate reduction in the frontal cortex (upper right); compared with FDG-PET of a healthy person (lower right). Reduction of activity in the heads of the caudate nuclei and frontal cortex was confirmed by voxel-based testing vs a healthy control group, demonstrating statistical significance at the uncorrected α = 0.01 level (lower left; brighter colors indicate lower p values). (C) Binding of patient 1 serum (1:100 dilution) to DPPX-transfected HEK293 cells. Healthy control serum (1:10 dilution) and HEK293 cells transfected with empty vector served as controls. (D) Primary hippocampal neurons were incubated for 2 hours with patient 1 serum (1:100 dilution), fixed, and double-stained for human immunoglobulin G (IgG) and DPPX using a commercial anti-DPPX antibody. The merge demonstrates overlap of both signals (confocal images). IVIG = IV immunoglobulin; n.d. = not detectable.

Figure 2. Anti–dipeptidyl-peptidase-like protein 6 sera cause hyperactivity of enteric nervous system neurons

(A) Indirect immunofluorescence of mouse small intestine with purified patient 1 immunoglobulin G (IgG) (1:100) or control serum (1:100) demonstrates staining of the myenteric plexus by patient IgG. Costaining for microtubule-associated protein 2 (Map2) confirms binding of patient IgG to neuronal structures; nuclei are counterstained in blue (4′,6-diamidino-2-phenylindole [DAPI]). (B) Sections of monkey (Macaca mulatta) gut were incubated with patient 1 or control serum (1:10), revealing staining of the myenteric plexus by the patient's serum. (C) Indirect immunofluorescence of guinea pig ileum whole mount preparations of submucous and myenteric plexus with serum of patient 1 (1:1,000) shows strong staining of neuronal structures. (D) Enteric nervous system neurons of guinea pig myenteric plexus were loaded with the voltage sensitive dye DI-8-ANEPPS and electrical activity was recorded as changes in fluorescence intensity (% ∆F/F) after local application of patient or control serum for 200 ms (horizontal black bar). Top trace shows no response to the control serum. Second trace shows 2 action potentials (spikes) after application of serum from patient 1. Similar results were obtained with serum from another patient with anti–dipeptidyl-peptidase-like protein 6 (DPPX) encephalitis (patient 2). Scale bars apply to all traces. (E) Same experimental setting as in (D) using human submucous plexus. The response to the application of patient sera consists of bursts of action potentials that last throughout the recording period. (F) Percentage of guinea pig and human enteric nervous system neurons that fired action potentials following application of patient or control serum. Numbers above the bars indicate the absolute numbers of responding neurons out of the total number of neurons analyzed. **p < 0.001; χ²-test (guinea pig), McNemar test (human). (G) Frequency of action potentials in guinea pig and human enteric nervous system neurons following application of anti-DPPX or control sera. Numbers of neurons analyzed are indicated above the bars. *p < 0.05, **p < 0.001; guinea pig, Mann-Whitney U test; human, Wilcoxon test (patient 1), paired t test (patient 2). AP = action potential; cm = circular musculature; lm = longitudinal musculature; mp = myenteric plexus; smp = submucous plexus.

Figure 3. Staining of CNS neurons by anti–dipeptidyl-peptidase-like protein 6 serum

(A) Indirect immunofluorescence of rat cerebellum with patient 1 and control serum (both at 1:10) demonstrates prominent staining of the cerebellar cortex, particularly in the granule cell layer glomeruli, by the patient's serum. (B) Indirect immunofluorescence of murine hippocampus sections with patient 1 and control serum (both at 1:100) reveals strong immunoreactivity within the neuropilar regions of the cornu ammonis and dentate gyrus. (C) Staining of living cultured primary hippocampal neurons with patient 1 serum, purified patient 1 immunoglobulin G (IgG), or control serum (all at 1:100) showed a punctuate staining pattern at the cell surface following incubation with patient serum or purified IgG. (D) Hippocampal cultures were incubated with patient 1 serum as before, fixed and counterstained for microtubule-associated protein 2 (Map2) as somatodendritic marker. Immunoreactivity of the patient's serum shows a clear somatodendritic distribution. The boxed areas labeled a and b in the merged image are also shown in higher magnification. All microimages were obtained by confocal imaging. CA = cornu ammonis; DG = dentate gyrus; gcl = granule cell layer; ml = molecular layer; pcl = Purkinje cell layer; Slu = stratum lucidum.

Figure 4. Binding of anti–dipeptidyl-peptidase-like protein 6 serum to excitatory and inhibitory synapses and association with Kv4.2

Cultured hippocampal neurons were incubated for 2 hours with patient 1 serum (1:100), fixed, and double-stained for the general synaptic marker synapsin (A), the vesicular glutamate transporters 1 and 2 (VGLUT1/2, B), or the vesicular GABA transporter (VGAT, C) to mark excitatory and inhibitory synapses, respectively. The patient's serum reveals a predominant, though not exclusive, synaptic staining pattern, including both glutamatergic and GABAergic synapses. (D) Hippocampal neurons were incubated for 2 hours with purified patient 1 immunoglobulin G (IgG) (1:100), fixed, and double-stained for human IgG and Kv4.2. Both signals showed a close colocalization at neuronal surfaces. The boxed areas labeled a and b in the merged image are also shown in higher magnification.

Figure 5. Antibody-mediated downregulation of dipeptidyl-peptidase-like protein 6 and Kv4.2 from neuronal membranes

(A) Membrane fractions were generated from cultured hippocampal neurons and processed for immunoblotting after preincubation of neurons with patient 1 or control immunoglobulin G (IgG) for 3 days (1:100, daily application). Immunoblots were developed with commercial anti–dipeptidyl-peptidase-like protein 6 (DPPX), Kv4.2, and Na⁺/K⁺-ATPase antibodies. Glycerinaldehyde-3-phosphate dehydrogenase (GAPDH) was used as loading control. (B) Quantification of DPPX, Kv4.2, and Na⁺/K⁺-ATPase expression in relation to the GAPDH signal and normalized to untreated cultures. Data are means ± SEM from 3 (DPPX, Kv4.2) or 2 (Na⁺/K⁺-ATPase) independent experiments. (C) Membrane fractions were generated from cultured hippocampal neurons and processed for immunoblotting after preincubation of neurons with patient 1 or control serum for 3 days (1:100, daily application). Fractions were tested for DPPX expression using a commercial antibody. Actin was used as loading control. (D) Quantification of DPPX expression in relation to the actin signal, normalized to untreated cultures. Data are means ± SEM from 3 independent experiments. Statistical significance was assessed by Mann-Whitney U test.