Appendix e-1

Case report (cerebellar degeneration and antibodies to mGluR1)

In 2007 a 69-year-old man noted unsteady gait and speech problems. Over the next several months, he developed cerebellar ataxia of the upper and lower extremities, with rapidly progressive decline in his speech and ability to walk. He lost the ability to sit straight or walk without assistance. In addition, he had nystagmus and difficulty directing and maintaining fixation of gaze. He was treated with intravenous methylprednisolone (1 g daily for 3 days) every 4 weeks for 5 cycles, then every 12 weeks for 3 more cycles. There was a transient improvement of symptoms (score in Scale for the Assessment and Rating of Ataxiae3 improved from 19 to 14), but subsequently symptoms worsened and stabilized at score of 17.5. Since then, symptoms have not changed substantially despite monthly treatments with intravenous methylprednisolone. At the last follow-up in January 2011, he had severe dysarthria and disabling limb, truncal, and ocular ataxia.

Brain MRI at symptom onset showed signs compatible with small vessel ischemic disease. Follow-up MRI 1 year later showed cerebellar atrophy. Initial CSF studies demonstrated 8 white blood cells /µl (predominantly lymphocytes; normal <4/ µl), normal glucose and protein levels, and negative findings for viral (HSV, VZV PCR) or bacterial infections. Antibodies to onconeuronal antigens, glutamic acid decarboxylase (GAD), and cell surface neuronal antigens (NMDA, AMPA, GABA(B), glycine receptors, and LGI1 and CASPR2 proteins) were all negative. Genetic testing for the following conditions was negative: spinocerebellar ataxia (SCA types 1, 2, 3, 6, 7, and 17), Friedreich’s ataxia (FRDA), and Fragile-X tremor-ataxia syndrome (FXTAS). CT of chest, abdomen and pelvis, whole-body PET scan, and ultrasound studies of the testes and prostate were all negative for cancer.

The patient’s CSF and serum contained antibodies that reacted with the neuropil of rodent brain, predominantly the molecular layer of the cerebellum (Figure 1N). Studies using HEK293 cells transfected with mGluR1 or mGluR5 showed that patient’s antibodies reacted with mGluR1, but not with mGluR5 (Figure e-2 and Figure 1).

Groups of patients screened for antibodies against mGluR5 and mGluR1

In order to examine the occurrence of mGluR1 and mGluR5 antibodies in various patient populations, we screened sera (1:200) and/or CSF (1:5) from the following groups of patients for reactivity with HEK293 cells transfected to express mGluR5 or mGluR1: 11 patients with HL and encephalitis (none of them with limbic encephalopathy or Ophelia syndrome); 12 patients with HL and anti-Tr associated cerebellar degeneration; 3 patients with HL and other syndromes (ataxia or neuropathy); 18 patients with encephalitis without cancer but with “unclassified” anti-brain antibodies (negative for NMDA, AMPA, GABA-B receptor, and LGI1 and Caspr2 proteins), and 60 patients with one of the following disorders, viral encephalitis, multiple sclerosis, Rasmussen’s encephalitis, or onconeuronal antibody associated paraneoplastic disorders. Studies were approved by the University of Pennsylvania Institutional Review Board and all patients or their surrogates gave informed consent.

Methods

Brain and neuron immunohistochemistry

Rodent brain sections were obtained as described previously.e4 Brains were rapidly removed, sagitally split, and fixed in 4% paraformaldehyde for 1 hour, then cryopreserved in 40% sucrose for 24 hours, embedded, and sagitally sectioned. Immunostaining with patients’ sera (dilution 1:200) was performed as previously reported and developed with the avidin-biotin-peroxidase method.e4

Hippocampal neuronal cultures were obtained as described.e5 Neurons were incubated with patient or control serum (1:200) for 1 hour at room temperature followed by a fluorescent FITC-conjugated goat anti-human IgG secondary antibody (A-11014, Molecular Probes, Eugene, OR; 1:2000) following a method described previously.e6

Immunoprecipitation and immunoblot

Live hippocampal neurons were used for immunoprecipitation as described previously.e6 Distinctive protein bands precipitated by patient’s serum were excised from the Coomassie blue stained gel and analyzed using mass spectrometry at the proteomic facility at the University of Pennsylvania. After characterization of the antigen, aliquots of the immunoprecipitate were transferred to nitrocellulose and blotted with a rabbit antibody to mGluR5 (ab27190, Abcam, Cambridge, MA; 1:1000). The reactivity was developed using the appropriate biotinylated secondary antibodies (1:2000) and the avidin-biotin peroxidase, diaminobenzidine method.

HEK-293 cell based assays

HEK-293 cell based assays were performed as reported.e6 In brief, HEK-293 cells were plated on poly-D-lysine coated 10 mm coverslips and allowed to grow for 24 hours, then transfected with lipofectamine 2000 (2 µL/coverslip; Invitrogen, Carlsbad, CA) and plasmid DNA for mGluR5 or mGluR1-EGFP (2 ng plasmid DNA / coverslip). After 24 hours, coverslips were fixed with 4% paraformaldehyde for 10 minutes, washed with phosphate buffered saline (PBS), permeabilized with 0.3% triton X-100, washed with PBS, and blocked with 5% goat serum for 1 hour. Patient or control serum (1:200) or CSF (1:5) along with a rabbit antibody to mGluR5 (ab27190, Abcam, Cambridge, MA; 1:1000) were applied for 1 hour, then the coverslips were washed with PBS and labeled with the appropriate fluorescent secondary antibodies (A21206, donkey anti-rabbit IgG, Invitrogen, and A11014, goat anti-human IgG, Molecular Probes, Eugene, OR; both at 1:1000). Since cells expressing mGluR1-EGFP were fluorescent, a commercial antibody to mGluR1 and corresponding secondary antibody were not needed for our assay for mGluR1 antibodies. Results were captured by a fluorescence microscope using Zeiss Axiovision software (Zeiss, Thornwood, NY, USA).

mGluR5-null mice

mGluR5 knockout mice were bred and genotyped as described previously.e7 Brains of mGluR5-null and control mice were processed and labeled as described above. To examine the distribution of mGluR5 in mouse brain and to verify the deletion of mGluR5 in brains of mGluR5-null mice, brain sections from wild-type and mGluR5-null mice were labeled with a rabbit antibody against mGluR5 (06-541, Milipore, Billerica, MA; 1:500). This reactivity was developed using the appropriate biotinylated secondary antibodies (1:2000) and the avidin-biotin peroxidase, diaminobenzidine method. The reactivity of patients’ sera with mGluR5-null and control mice was determined as indicated above.

e-References

e1.Medeiros LJ, Greiner TC. Hodgkin's disease. Cancer 1995;75:357-369.

e2.Lancaster, E., Martinez-Hernandez, E., Dalmau, J. Encephalitis and antibodies to synaptic and neuronal cell surface proteins. Neurology, in press.

e3.Schmitz-Hübsch T, du Montcel ST, Baliko L et al. Scale for the assessment and rating of ataxia: development of a new clinical scale. Neurology 2006; 66: 1717-20.

e4.Ances BM, Vitaliani R, Taylor RA, et al. Treatment-responsive limbic encephalitis identified by neuropil antibodies: MRI and PET correlates.Brain 2005; 128: 1764-77.

e5.Buchhalter JR, Dichter MA. Electrophysiological comparison of pyramidal and stellate nonpyramidal neurons in dissociated cell culture of rat hippocampus.Brain Res Bull 1991; 26:333-8.

e6.Lancaster E, Lai M, Peng X, et al. Antibodies to the GABA(B) receptor in limbic encephalitis with seizures: case series and characterisation of the antigen.Lancet Neurol 2010; 9: 67-76.

e7.Xu J, Zhu Y, Contractor A, et al. mGluR5 has a critical role in inhibitory learning.J Neurosci 2009; 29: 3676-84.