Supplementary webappendixThis webappendix formed part of the original submission and has been peer reviewed. We post it as supplied by the authors.

Supplement to: Sabater L, Gaig C, Gelpi E, et al. A novel non-rapid-eye movement and rapid-eye-movement parasomnia with sleep breathing disorder associated with antibodies to IgLON5: a case series, characterisation of the antigen, and post-mortem study. Lancet Neurol 2014; published online April 3. http://dx.doi.org/10.1016/S1474-4422(14)70051-1.

This supplementary webappendix has been corrected. The corrected version first appeared at thelancet.com/specialty on Dec 8, 2014

1

Appendix Table of Contents Methods Description of clinical cases Neuropathological studies Table 1S: Primary antibodies used in the neuropathological analysis Table 2S. Topographical distribution of neuronal loss and tau pathology Table 3S: The IgLON family Figure 1S: IgG Isotype analysis of antibodies against IgLON5.

Figure 2S: Identification of IGLON5 protein by mass spectrometry. Figure 3S: Immunoblot of HEK cells transfected with GFP-tagged IgLON5. Figure 4S: Immunoabsorption with IgLON5 Figure 5S: Supratentorial Tau-pathology Figure 6S: Abnormal tau deposits and neurofibrillary tangles. Figure 7S. IgLON5 immunohistochemistry in human brain tissue References Methods Control groups Serum or CSF of 298 patients were used as controls including, 45 with pathologically confirmed Alzheimer disease, 32 with clinical diagnosis of progressive supranuclear palsy (PSP), 22 with Idiopathic Parkinson disease, 21 with DNA-binding protein 43 (TDP-43) frontotemporal dementia, 18 with multiple system atrophy, 13 with diffuse Lewy body disease, 8 with corticobasal degeneration, 25 with idiopathic RBD, 28 with hypocretin deficient narcolepsy, 54 with multiple sclerosis, and 32 with anti-Lgi1 encephalitis. Samples were selected without knowledge of clinical details except for patients with PSP, frontotemporal dementia, multiple system atrophy, diffuse Lewy body disease, and corticobasal degeneration that were used all the samples available. Video-Polysomnographic studies Nocturnal V-PSG included electrooculography, electroencephalogram (EEG) (F3, F4, C3, C4, O1 and O2, referred to combined ears), submental electromyogram (EMG), surface bilateral EMG of the flexor digitorum superficialis in the upper limbs, and anterior tibialis and extensor digitorum brevis in the lower limbs. Electrocardiography, nasal and oral airflow, thoracic and abdominal movements, and oxyhemoglobin saturation were also recorded. Immunohistochemistry on rat brain, HEK293 cells, and neuronal cultures

Female Wistar rats were euthanized and the brain was removed, sagittally sectioned, immersed in 4% paraformaldehyde at 4°C for 1 hour, cryoprotected with 40% sucrose for 24 hours, and snap frozen in chilled isopentane. Immunohistochemistry using a standard avidin-biotin peroxidase method was applied using patients’ serum (diluted 1:200) or CSF (1:5), followed by the appropriate secondary antibody, as reported.1 To study the distribution of IgG subclasses of the antibody, the same immunohistochemistry technique was used changing the secondary antibody by biotinylated mouse monoclonal antibodies to human IgG 1-4 subclasses (Sigma, St. Louis, MO) (dilutions: anti-IgG1 1:100, anti IgG2 1:200, anti-IgG3 1:200, and anti-IgG4 1:200) or to human IgM (Southern Biotechnology Associates, Inc, Birmingham, AL, USA) as described.2

To show if anti-IgLON5 antibodies of different patients recognized similar epitopes, rat brain sections were pre-incubated with undiluted anti-IgLON5-positive serum for three hours followed by a biotinylated IgG obtained from another positive anti-IgLON5 serum, in 10% normal human serum, overnight at 4°C, and the Vectastain Elite ABC complex (Vector Labs, USA) for 40 min. The reaction was developed with 0.05% diaminobenzidine with 0.01% hydrogen peroxide in phosphate-buffered saline

2 (PBS) with 0.5% Triton X-100. As controls, sections were incubated with biotynilated IgG from a normal human serum.

Transfected HEK293 cells with the GFP-tagged IGLON clones were incubated with patients’ serum (1:40) or CSF (1:2) for 2 hours at room temperature, fixed in 4% paraformaldehyde, permeabilized with 0.3% Triton X-100 and the corresponding fluorescent secondary antibody (Alexa Fluor from Invitrogen). Results were photographed under a fluorescence microscope using Zeiss Axiovision software.

Rat hippocampal neuronal cultures were prepared as reported.3 Fourteen days live neurons grown on coverslips were treated for 1 hour at 37º C with patients’ or control serum (final dilution 1:200) or CSF (1:5). After removing the media and extensive washing with PBS, neurons were fixed with 4% PFA, and incubated with anti-human IgG Alexa Fluor secondary antibody diluted 1:1000 (Molecular Probes, OR). Results were photographed under a fluorescence microscope using Zeiss Axiovision software (Zeiss, Thornwood, NY).4

Immunoprecipitation and mass spectroscopy

Immunoprecipitation experiments were done with cultures of rat hippocampal neurons grown in 100mm wells, and incubated at 37°C with patients’ or control serum (diluted 1:100) for 1 hour. Neurons were then washed with PBS, lysed with buffer containing protease inhibitors (P8340; Sigma Labs, St. Louis, MO, USA), and centrifuged at 16,000 g for 20 minutes at 4°C. The supernatant was retained, incubated with protein A/G agarose beads (20423; Pierce, Rockford, IL) overnight at 4°C, and centrifuged. The pellet was resuspended in Laemmli buffer, boiled for 10 minutes, separated in a 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis, and the proteins visualized with EZBlue gel staining (G1041; Sigma Labs). Because the EZBlue gel staining did not identify specific protein bands, gels were cut into ten slices and sent for mass spectrometry to the Proteomics Core Facility at the University of Pennsylvania. Protein bands were trypsin digested and analyzed with a nano liquid chromatography (nano LC)/ nanospray/ linear ion trap (LTQ) mass spectrometer (Thermo Electron Corporation, San Jose, CA) as reported.5 The Xcalibur software (Thermo Scientific, Waltham, MA) was utilized to acquire the raw data and Sequest program (ThermoFinnigan, San Jose, CA; version SRF v. 5) to match the results with the UniProtKB/Swiss-Prot protein sequence database. The Scaffold 3.3 program was used to analyse the files generated. Protein identifications were accepted if they could be established at greater than 95.0% probability and contained at least three identified peptides.

Clinical description of the eight patients with IgLON5 antibodies Patient 1 (index case)

A 59 year-old man was referred to our sleep unit with a 18 month history of abnormal sleep behaviors. The patient had been diagnosed of obstructive sleep apnea syndrome (apnea-hypopnea index: 81 in a PSG) in another center but continuous positive airway pressure therapy (CPAP) failed to improve the symptoms except the snoring. His wife reported that just after falling asleep he “snored” loudly and presented frequent vocalizations and limbs movements. He was constantly talking and groaning. Sometimes he laughed or cried. He was continuously jerking, punching, or kicking. Often he gesticulated and mimicked activities of his daily living such as working or eating. The patient was unaware of his behaviors and dreaming was absent. His wife, however, had often the impression that he was dream-enacting, like arguing, discussing or working. Sleep fragmentation and occasional episodes of intense sleepiness were also present.

Associated symptoms included a continuous inner feeling of restlessness resembling akathisia. This restlessness was sometimes focused in the abdomen and was often relieved by intense exercise but no by walking or stretching the legs and did not improve with pramipexole 0.18 mg/day. Other complaints were mild swallowing difficulties, excess of salivation and drooling, an ill-defined abdominal discomfort that often overlapped with the akathisia-like restlessness, urinary urgency with hesitation, nocturia, erectile dysfunction, and occasional episodes of sudden an intense sweating. Fatigue, apathy depressed mood and mild memory and attention difficulties also occurred. As a consequence of all these symptoms, the patient was unable to work. Since depression was suspected, antidepressants, hypnotics and neuroleptics were introduced with worsening of sleep-related behaviors and development of nocturnal episodes of confusion.

When referred to our sleep unit, the neurological examination was normal. The scores of the neuropsychological evaluation (Auditory verbal learning test, Boston naming test, digit span, trail making A and B, Poppelreuter test, FAS word fluency test, and semantic fluency test) were within normal limits Routine blood and CSF analysis, brain MRI, EEG, and body CT were normal. Mutations in the prion

3 protein were absent. An EMG ruled out neuromyotonia and other neuromuscular disorders. Intravenous (IV) methylprednisolone (1 gram/day over 5 days) followed by oral prednisone (1 mg/Kg day during 1 month) resulted in mild improvement of sleep fragmentation, and abnormal sleep movements and behaviors. A second treatment of IV immunoglobulins (2g/kg over 5 days) and cyclophosphamide (1 gram, 3 doses 1 month apart) did not result in additional improvement. Over the ensuing 2 years (4 years after disease onset), patient’s sleep disturbances and associated symptoms have remained unchanged. Three video-PSG performed with the CPAP mask at 12 cm H2O during this period have shown persistence of the NREM-REM parasomnia. In another video-PSG without CPAP mask, however, stridor with associated obstructive sleep hypopneas and apneas (apnea-hypopnea index: 37) were recorded together with the NREM-REM parasomnia. A laryngoscopy showed mild right vocal cord paresis with swallowing difficulties and accumulation of saliva in the hypopharynx. Patient 2

A 55 year-old man was referred to our sleep unit with persistent excessive daytime sleepiness and abnormal movements and behaviors during sleep despite CPAP therapy. Two years before he had progressively developed an intense “snoring” while asleep with frequent jerks, movements and behaviors, nocturnal enuresis, sleep fragmentation and mild passive daytime somnolence. He was diagnosed of obstructive sleep apneas (apnea-hypopnea index: 59) in another center. Treatment with CPAP at 7 cm H2O eliminated “snoring” and apneas, but sleep movements and behaviors and excessive daytime sleepiness persisted.

His wife reported that he was continuously talking and moving while asleep. His speech was usually unintelligible but sometimes he called his father or son. Occasionally he shouted. Movements during sleep included frequent jerks, knocks, kicks and gesticulations. His wife had often the impression that he was dream-enacting, as arguing with someone. The patient was not aware of these behaviors and he did not recall any dreams. In addition, there were occasional nocturnal episodes of confusion in which he awoke disoriented and used the toilette unsuitably or opened the windows or doors. These episodes of nocturnal confusion dramatically worsened after introduction of tricyclic antidepressants. He also complained of mild intermittent dysphagia and drooling, which was particularly problematic at night. He had often to take off the CPAP mask and spit the excess of saliva. He complained of memory difficulties. Other symptoms included apathy, depressed mood, and an inner feeling of restlessness resembling akathisia.

Neurological examination was normal. The neuropsychological evaluation (Auditory verbal learning test, Boston naming test, digit span, trail making A and B, Poppelreuter test, FAS word fluency test, and semantic fluency test) was compatible with minor impairment of verbal episodic memory, decreased attention span, and deficit in visuospatial functions. Extensive blood analysis, EEG, EMG, brain MRI and cerebral (18) F-fluoro-2-deoxyglucose PET were also normal. CSF showed no cells and increased protein level (55 mg/dl; normal 15-45 mg/dL). Video-PSG with CPAP at 7 cm H2O demonstrated a NREM-REM parasomnia.

During four years the symptoms fluctuated, with periods lasting weeks of increased intensity. Multiple antidepressants and benzodiazepines were of no benefit. Three additional video-PSGs were performed and NREM-REM parasomnia persisted without significant changes. The last video-PSG was performed without CPAP mask. The intense “snoring” was found to be stridor with frequent associated obstructive sleep hypopneas and apneas (apnea-hypopnea index: 20). Laryngoscopy during wakefulness, however, was normal.

Treatment with IV methylprednisolone (1 gram/day over 5 days) and cyclophosphamide (1 gram, 3 doses 1 month apart) did not result in any improvement. The patient developed spontaneous multiple cutaneous hematomas with enlarged activated partial thromboplastin time (aPTT). Acquired hemophilia due to antibodies against clotting factor VIII was diagnosed. After three months of therapy with oral prednisone (1 mg/Kg day) and cyclophosphamide (50 mg/day), aPTT and levels of factor VIII normalized. One month later, the patient died suddenly while asleep with the CPAP mask. Autopsy study is detailed below. Patient 3

A 53 year-old male with obstructive sleep apnea syndrome was referred to our sleep unit with suspected REM sleep behavior disorder. One year before, intense episodic somnolence, mild sleep onset insomnia and abnormal sleep movements and behaviors developed in less than 3 months. While asleep he “snored” heavily and frequently talked (usually incomprehensibly), groaned and moved. He often punched, kicked, jerked, knocked or gesticulated. Dream recall was absent, but the patient’s wife reported that sometimes he seemed to be acting out of his dreams. A PSG was performed in another center. Frequent apneas were recorded (apnea hypopnea index: 33/h) and obstructive sleep apnea syndrome was

4 diagnosed. Although CPAP at 9 cm H2O eliminated apneas and the “snoring”, somnolence and sleep movements and behaviors persisted.

During this time, the patient also presented a restlessness resembling akathisia. This restlessness was an inner-feeling that was present during the whole day, without nocturnal worsening, and failed to improve with dopaminergic agonists. In addition, he developed mild dysphagia with occasional choking, nocturnal drooling, mild dysarthria, vague abdominal discomfort, urinary hesitance with poor stream, and mild unsteadiness with infrequent falls. His mood was low and he was apathetic and complained of mild attention and memory problems. Neurological examination was normal except for mild distal upper and lower limb choreic movements. The neuropsychological evaluation (Auditory verbal learning test, Boston naming test, digit span, trail making A and B, Poppelreuter test, FAS word fluency test, and semantic fluency test) was compatible with minor impairment of verbal episodic memory, decreased attention span, and deficit in visuospatial functions.

General blood and CSF analysis, brain MRI, dopamine transporter SPECT, and EMG were normal. A video-PSG with his CPAP mask was performed in our center and showed a NREM-REM parasomnia without apneas. During the following 4 years his symptoms were basically unchanged. The NREM-REM parasomnia persisted in several video-PSG studies despite treatment with clonazepam and melatonin. As a consequence of these symptoms, he was unable to work. A new video-PSG without CPAP showed stridor with frequent obstructive sleep apneas (apnea hypopnea index: 44). Laryngoscopy showed mild bilateral vocal cord paresis. Treatment with IV methylprednisolone (1 gram/day over 5 days) and cyclophosphamide (1 gram, 3 doses 1 month apart) did not result in any improvement. Patient 4

A 69 year-old woman presented insomnia with fragmented sleep along with anxiety and memory, attention, and planning difficulties for a few weeks. Episodes of syncope and generalized involuntary dyskinetic movements were also present. In addition she developed bouts of irresistible daytime sleep and loud snoring with frequent sleep-talking and sleep movements that included continuous rocky and jerky limb movements, punches, kicks and some gesticulations. Nightmares were absent. Sleep apneas were documented in a PSG but CPAP was not tolerated.

Neurological examination showed cognitive deterioration (MMSE:21), dysarthria, choreic upper limb movements with orofacial dyskinesias and mild vertical upward and downward gaze limitation with normal coordination, gait and postural reflexes.

Routine blood and CSF analysis, brain MRI, 18-FGD body PET, analysis for prion gene mutations, were normal or negative. Electrocardiogram disclosed a bradiarrhythmia, The EEG showed mild diffuse slowing. The patient did not benefit of intravenous high dose steroids. During the following year her cognitive problems worsened progressively and confusional episodes appeared. Episodes of central hypoventilation occurred and several months later, she suddenly died while awake.

Patient 5

A 71 year-old woman consulted because of 12 years history of slowly progressive balance and gait difficulties followed by abnormal sleep movements. At the age of 59 years, she noticed unsteady gait with occasional falls that slowly progressed over the ensuing 8 years. At age of 67, she started to “snore” loudly and frequently talked and moved her arms and legs during sleep. Dreaming was absent. One year later, her voice became hoarse and hypophonic. At age of 69, she developed diplopia with horizontal gaze palsy and gaze-evoked nystagmus to the left and upward, choreic limb movements, dysphagia, and nocturia. Over the next year her gait and balance worsened considerably. Mild excessive daytime sleepiness was present. She presented episodes of evening disorientation and nocturnal confusion with agitation, loud shouting and fear or odd behaviors (e.g recite poems while urinating in the hallway or clean the bathroom during the night). Urinary urgency and incontinency were also present.

At age of 70, neuropsychological evaluation (block span test, digit span test, word pairs memory test, color word test, trail making test A + B, Colored progressive matrices (A/B), Regensburg word fluency test) disclosed impairment in non-verbal episodic memory, slowed cognitive speed and reduced selective attention. The neurological examination demonstrated that gait was wide-based and impossible without assistance. Postural reflexes were severely impaired. There was mild dysarthria, limb ataxia and hypokinesia. Choreic movements were observed in the face and distal limbs.

General blood analysis was normal except for an IgM kappa monoclonal gammopathy of unknown significance. Acanthocytosis and Wilson's disease were ruled out. Genetic testing for spinocerebellar ataxias and Huntington disease was negative. Cranial MRI revealed mild atrophy of the brainstem and cerebellum. CSF analysis showed no abnormalities except for positive IgG oligoclonal bands. EEG and

5 EMG were normal. A video-PSG demonstrated stridor, obstructive apneas-hypopneas (apnea hypopnea index: 84/h) with frequent vocalizations and movements. IV methylprednisolone (500 mg/day over 5 days) continued with an oral dose of 60 mg daily for two months and later rituximab resulted in no improvement. She made a sudden death at home while she was walking to go to the bathroom. Patient 6

A 63 year-old female with bronchial asthma consulted because of a 5- year history of gait unsteadiness. Her gait problems had progressed slowly and in the last year falls became frequent and gait was impossible unaided. Over the last 3 years, she also developed blurred vision, mild dysphagia with marked drooling, and mild involuntary dyskinetic perioral and limb movements. In addition, she presented obstructive sleep apnea and stridor diagnosed be means of PSG, but CPAP treatment was not tolerated. Nocturnal sleep was fragmented with nocturia and she complained of frequent nightmares.Her relatives reported frequent limb movements and jerks during sleep. During the last year, she complained of mild memory, attention, planning difficulties, apathy, and depressed mood. She consulted several neurologists that gave the tentative diagnosis of sporadic adult onset ataxia, gluten ataxia, and multiple system atrophy.

Neurological examination demonstrated a mini-mental state score of 28/30. A battery of neuropsychological tests was normal (ADAS test, Boston naming test, digit span, trail making A and B, Stroop test, Brixton test, Raven test, word fluency test, Buschke memory test). Postural reflexes were impaired and her gait was unsteady, wide-based, and impossible unaided. There was dysarthria and mild limb dysmetria. Oculomotor examination disclosed frequent saccadic intrusions on pursuit and gaze-evoked vertical and horizontal nystagmus. She presented choreic movements in the face and distal limbs. The rest of the neurological examination was normal.

Routine blood analysis, vitamin E and ceruloplasmin levels, genetic testing for spinocerebellar ataxias, CSF analysis (with PCR for Tropheryma whipplei), EEG, and EMG were normal. Brain MRI showed mild brainstem and cerebellar atrophy. Spinal MRI was normal. Antibodies to cardiolipin and gliadin were positive. Patient did not improve with two cycles of intravenous immunoglobulins (0.4 grams/kg/d X 5 days). She developed episodes of central hypoventilation that required admission to the intensive care unit and intubation. She died during attempts to withdraw the intubation. Patient 7

A 76 year-old female was admitted to the emergency department because of a two-week history of gait difficulties with falls. Mild speech difficulties and dysphagia were also present. Neurological examination showed loss of balance with gait failure. Postural reflexes were severely impaired and she was unable to stand unsupported. She could barely walk even with the help of a person and had a short-stepped and slightly wide-based gait. Additional findings included saccadic intrusions on pursuit eye movements and mild dysarthria. Two days after admission, she developed sudden respiratory failure with central hypoventilation that required admission in the intensive care unit with intubation and ventilation. Only when asked about her sleep, her husband reported a 4-month history of loud respiratory noise with frequent sleep vocalizations and jerky movements. She was unaware of these sleep disturbances and nightmares were absent. A video-PSG performed in the ICU after ventilation weaning showed a reduced sleep efficiency (26%) due to noise, light and continuous stimulation by ICU nurses, but during sleep a NREM parasomnia was recorded. REM sleep was not recorded and stridor and apneas were not observed. A laryngoscopy confirmed bilateral vocal cord paresis. Routine blood and CSF analysis, brain MRI, EMG, and thoracic CT were normal. IV methylprednisolone (1 gram/d over 5 days) followed by oral prednisone (1 mg/Kg) improved respiratory function and sleep movements. A new video-PSG, however, showed stridor with obstructive apneas (apnea-hypopnea index: 23), brief periods of REM sleep in the form of RBD, and NREM sleep without vocalizations or movements. She had two additional episodes of hypoventilation and tracheotomy was performed. IV cyclophosphamide (1 gram, 2 doses 1 month apart) resulted in mild improvement in balance and gait and the patient was able to walk with a walker. A new video-PSG a month later showed that NREM-REM sleep parasomnia had recurred with persistent stridor. Five months after admission, after improvement of respiratory function, the tracheotomy was closed and she was discharged to a nursing home. One month later she died suddenly during sleep. Autopsy study is detailed below. Patient 8

A 65 year-old woman with a long lasting history of severe depression was admitted because of a few weeks history of unsteady gait and frequent falls. During this period, the patient had also developed sleep problems, mild memory and planning difficulties with apathy, worsening of her depressed mood, diplopia, dysphagia with choking and drooling, dysarthria, urinary urgency and incontinence, and

6 episodes of mandibular and hemifacial spasms lasting less than 1 minute. Sleep problems consisted in loud “snoring with fragmented sleep and abnormal sleep movements and vocalizations with jerking, kicking and more complex behaviors. Neurological examination showed a normal cognitive status. Saccadic eye movements were slow with limitation of upward gaze in the left eye and bilateral gaze-evoked horizontal and vertical nystagmus. There was a mild dysarthria and dysmetria in the four limbs. Deep tendon reflexes were increased with bilateral ankle clonus but plantar responses were flexor. She had severe impairment of postural reflexes and had a wide-based gait that was only possible when supported by another person. The patient developed a sudden episode of central hypoventilation requiring intubation. Episodes of abnormal sleep movements and behaviors were witnessed during the ICU admission. PSG in the ICU showed frequent obstructive sleep apneas (apnea-hypopnea index: 97) that responded to CPAP therapy. However, abnormal sleep movements and behaviors persisted.

Routine blood and immunological analysis, CSF analysis, brain and spinal MRI, EMG, EEG, dopamine transporter SPECT, 18-FDG body PET, and body CT scan were normal. Episodes of hypoventilation disappeared and her gait significantly improved with intravenous immunoglobulins (0.4 gram /kg/d X 5 days) and methylprednisolone (1gram/d X 5 days) followed by rituximab 375 mg/week for 4 weeks. Her gait was possible unaided, presenting only mild unsteadiness on turnings. The patient was discharged at home and two days later she suddenly died shortly after awakening at the morning. Neuropathologic studies Patient 2 Unfixed brain weight was 1410g. There were no signs of atrophy. Histology revealed mild to moderate neuronal loss, astrogliosis and microglial activation in anterior and ventral parts of dorsomedial, but not posterior thalamus, hypothalamic nuclei, periaquaeductal grey matter, pontine tegmental area, and mild involvement of formatio reticularis and magnocellular nuclei of medulla oblongata (Table 2S). There were no inflammatory infiltrates or complement deposits in the affected areas. Neurofibrillary tangles were detected on hematoxylin and eosin stained sections in hippocampus CA1 sector and in brainstem mainly in caudal raphe. Immunohistochemistry revealed extensive hyperphosphorylated tau (AT8) pathology accentuated in brainstem and in anterior thalamic, hypothalamic nuclei and hippocampus (Figure 5, Table 2S). In the brainstem, abundant pretangles involving nearly all neurons of nuclei propii of basis pontis along with few neurofibrillary tangles were observed. These were predominantly, but not exclusively, immunoreactive for 4-repeat tau isoforms, and were strongly immunoreactive for phospho-specific anti-tau antibodies Thr181, Ser262, Ser396, Ser422. There was also prominent tau pathology in raphe neurons and less intensely in magnocellular nuclei of reticular formation. Interestingly, fine-granular tau immunoreactivity was also detected in Golgi cells and glomerula of cerebellar granule cell layer (not shown). There was no involvement of neocortical areas, basal ganglia or spinal cord. In hippocampus, AT8 threads, few neurofibrillary tangles detected by Gallyas silver impregnation were observed (Figure 6S) and frequent pretangles without obvious neuronal loss were detected in all CA sectors and in several granular neurons of fascia dentate (Figure 5S). Moreover, tau pathology was also detected in basal forebrain and suprachiasmatic/prehypothalamic region including septal nuclei, diagonal band and preoptic area. There were no grain-like structures and no glial pathology was observed. No concomitant abnormal protein deposits such as beta-amyloid, alpha-synuclein, TDP43 or alpha-internexin were detected. Patient 7

Unfixed brain weight was 1180 g. The brain showed mild, prefrontally accentuated atrophy along with mild atrophy of medulla oblongata. Neuronal loss and gliosis were detected mainly in zona incerta, hypothalamus, periaqueductal grey matter, mesencephalic and pontine tegmentum, medulla oblongata, and cervical spinal cord (Table 2S). There were no inflammatory infiltrates or complement deposits in the affected areas. Globose and flame-shaped neurofibrillary tangles were already detected in hematoxylin-eosin stained sections in locus coeruleus, formatio reticularis, magnocellular nuclei, and intermediolateral column of spinal cord. There were abundant hyperphosphorylated tau aggregates (AT8) mainly in hypothalamus, in prehypothalamic region and in brainstem areas, especially involving midbrain and pontine tegmentum, and most prominently in medulla oblongata severely involving nucleus ambiguus

7 and magnocellular nuclei of reticular formation (Table 2S, Figure 5). In these areas, neurofibrillary pathology showed strong immunoreactivity for phospho-specific anti-tau antibodies Thr181, Ser262, Ser396, Ser422. Tau aggregates were neuronal, in form of pretangles, tangles and neuropil threads, with a similar distribution of 3-repeat and 4-repeat tau isoforms. Beyond the brainstem, hyperphosphorylated tau aggregates were mild or absent, except for suprachiasmatic/prehypothalamic regions such as preoptic area, diagonal band and substantia innominata (Table 2S). There were no grain-like structures and no glial pathology was observed. There was a mild, concomitant beta-amyloid angiopathy involving frontal leptomeningeal vessels with few neuritic plaques in frontal cortex. No alpha-synuclein, FUS, polyQ or alpha-internexin protein aggregates were detected. Isolated Bunina bodies and TDP43-protein aggregates were found in brainstem and anterior horn neurons of spinal cord, along with very mild degeneration of lateral and anterior corticospinal tract.

8 Table 1S: Primary antibodies used in the neuropathological analysis

Antibody Antigen (clone) Company, city, country AT8 Phosphorylated tau at Ser202/Ser205 Thermo Scientific, Waltham, MA, USA RD3 3-repeat tau isoforms (8E6/C11) Millipore, Temecula, CA, USA RD4 4-repeat tau isoforms (1E1/A6) Millipore, Temecula, CA, USA Thr181 Phosphorylated tau at threonine 181 Calbiochem, La Jolla, CA, USA Ser262 Phosphorylated tau at serine 262 Calbiochem, La Jolla, CA, USA Ser396 Phosphorylated tau at serine 396 Calbiochem, La Jolla, CA, USA Ser422 Phosphorylated tau at serine 422 Calbiochem, La Jolla, CA, USA Aβ-amyloid Beta-amyloid protein (6F/3D) DAKO, Glostrup, Denmark α-synuclein Alpha-synuclein (KM51) Novocastra, Newcastle, UK ubiquitin Ubiquitin DAKO, Glostrup, Denmark p62 3/p62 lck ligand BD Transduction Laboratories, Franklin Lakes, NJ, USA TDP-43 TAR-DNA binding protein 43kD (2E2-D3) Abnova, Taipei, Taiwan FUS Fused in sarcoma Sigma, St. Louis, MO, USA α-internexin Class IV neuronal intermediate filament (2E3) Invitrogen, Carlsbad, CA, USA polyQ Polyglutamine expansions (MAB1574) Millipore, Temecula, CA, USA orexin Orexin B Millipore, Temecula, CA, USA GFAP Glial acidic fibrillary protein DAKO, Glostrup, Denmark NF Neurofilament 200kD (RT97) Novocastra, Newcastle, UK C9 Human Complement C9 neoantigen Hycult Biotech, Plymouth, PA, USA CD68 Macrophages/microglia (KP1) DAKO, Glostrup, Denmark CD3 Pan-T cell marker DAKO, Glostrup, Denmark CD20 B cell marker (L26) DAKO, Glostrup, Denmark IgLON5 IgLON5 cell adhesion molecule Abcam, Cambridge, UK

9 Table 2S. Topographical distribution of neuronal loss and tau pathology Patient 2 (table 1) Patient 7 (table 1)

Brain region neuronal loss tau Neuronal loss Tau

Neocortex 0 0 0 0

Hippocampus CA1, CA4 + ++ 0 +

Hippocampus CA2 0 +++ 0 0*

Dentate gyrus 0 ++ 0 0*

Entorhinal cortex + ++ 0 +

Amygdala 0 0* 0 +

Striatum 0 0 0 0*

Pallidum, external 0 0* 0 +

Pallidum, internal 0 + 0 ++

N. basalis Meynert + ++ 0 +

Substantia innominata + ++ + ++

Septal nuclei + ++ 0 +

Diagonal band + ++ + ++

Preoptic area + ++ + ++

Zona incerta 0 0* ++ ++

Subthalamic nucleus 0 0* + +

Thalamus

Anterior + ++ 0 0*

Dorsomedial + ++ + +

Posterolateral 0 0 0 0

Pulvinar 0 0 0 0

Hypothalamus

N. paraventricularis + + 0 +

N. supraopticus + + 0 +

N. ventromedialis + +++ + ++

N. tuberales + ++ + ++

N. posterior + ++ + ++

Corpus mamillare 0 0* 0 0*

Brainstem/cerebellum

N. laterodorsal tegmental + ++ + +++

N. pedunculopontine + ++ + +++

Periaqueductal grey ++ + + ++

Substantia nigra 0 0* 0 0*

10 Coeruleus/subcoeruleus complex 0 + 0 +

Central raphe (pons) + +++ + +

N. propii basis pontis 0 +++ 0 0

Dorsal n. vagal nerve + + + +

N ambiguus + ++ +++ +++

N. magnocellularis + ++ ++ +++

Inferior olives 0 0* 0 0*

Cortex cerebellum 0 ++ 0 0

Dentate nucleus 0 0* 0 0*

Cervical spinal cord 0 0* + +++

Scores: 0 = absent (e.g. Fig. 5D1 for tau immunoreactivity), + = mild (e.g. Fig. 5A1), ++ = moderate (e.g. Fig. D2) and +++ = severe (e.g. Fig. 5C2, E1). 0* = isolated neuropil threads at 200x or 400x magnification.

11 Table 3S. The IgLON family Gene Protein Predominant adult brain

expression Neuronal localization

IGLON1 Opioid binding protein/cell adhesion molecule-like (OBCAM)a

Cortex/hippocampus6 Postsynaptic dendritic and somatic synapses6

IGLON2 Neurotriminb Sensorimotor cortex, cerebellum8 Surface of axons and dendrites9

IGLON3 Limbic system-associated membrane protein (LAMP)a

Limbic system7 Surface of growth cones and postsynapsis in adult brain.7

IGLON4 Neural growth regulator 1 (NEGR1)a (KILON)b

Widespread6 Postsynaptic dendritic and somatic synapses6

IGLON5 IgLON5 Widespreadc Membrane surface a Human orthologue; bRodent orthologue; chttp://www.ebi.ac.uk/gxa/experiment/E-GEOD-803/ENSG00000142549?ef=.

12 Figures

Figure 1S: IgG Isotype analysis of antibodies against IgLON5.

Reactivity of a patient’s serum with rat hippocampus after incubation with antibodies specific for human IgG1 (A), IgG2 (B), IgG3 (C), and IgG4 (D). Robust neuropil immunostaining is only observed with IgG4. Scale bar=200 µm

Figure 2S: Identification of IGLON5 protein by mass spectrometry. Proteomic results of two independent immunoprecipitation experiments with rat hippocampal neurons. Upper panels: Tables containing the sequences of the predicted peptides (three in A, experiment 1 and seven in B, experiment 2) matching the fragmentation spectra after mass spectrometry analysis. The probability of the peptide identification, XCorr score and DCn score calculated by Sequest program is also included. Lower panels: The identified peptides are shown in red within the complete rat IGLON5 protein (Swiss Prot accession number, IPI00367494). (14% of protein coverage in A and 29% in B)

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Figure 3S:Immunoblot of HEK cells transfected with GFP-tagged IgLON5. Strips were incubated with an IgLON5 commercial antibody (lane 1) and serum from the 8 patients with IgLON5 antibodies detected by the cell based assay (lanes 2-9). Only the commercial antibody sowed a positive band indicating that IgLON5 antibodies of patients’ serum recognize conformational epitopes. Note that the molecular weight of the band recognized by the antibody is close to 64 KDa. that corresponds to IGLON5 predicted molecular weight protein (37 KDa) plus the molecular weight of the GFP tag (27KDa).

Figure 4S: Immunoabsorption with IgLON5 IgLON5 antibody-positive serum absorbed with HEK cells transfected with or without IgLON5. Only serum absorbed with HEK cells transfected with IgLON5 did not react with the neuropil of rat brain (B) and cultures of rat hippocampal neurons (D). IgLON5 reactivity was preserved when the serum was absorbed with non-transfected HEK cells (A, C). Scale bar A and B=1000 µm, C and D= 20 µm

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Figure 5S: supratentorial Tau-pathology Tau-pathology was absent in cortical areas in patient 7 (A1, frontal cortex) and in patient 2 (A2, frontal cortex), as well as in striatum of both patients (B1, patient 7; B2, patient 2). While hippocampus in patient 7 was only very mildly affected by few AT8 positive pretangles in CA4-CA1 and fascia dentata (C1, D1 and inset), in patient 2 abundant pretangles were detected in CA2 (D2) and CA4 sector and tangles, neuropil threads and pretangles in CA1 sector, subiculum and few in parahippocampal region (C2).

Figure 6S: Abnormal tau deposits and neurofibrillary tangles.

Detailed view of neurons of basis pontis of patient 2 shows single neurofibrillary tangles on HE stained sections (A, arrow), that are strongly argyrophilic as seen by Gallyas silver impregnation (B, arrow). In this staining, few fine neurites are also detected (C, arrows). Tau (AT8) immunohistochemistry shows widespread neuronal immunoreactivity (D) in form of pretangles (E), and a few neurofibrillary tangles (F).

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Figure 7S. IgLON5 immunohistochemistry in human brain tissue A1, B1: Immunohistochemistry with commercially available anti-IgLON5 antibodies reveals a fine synaptic immunoreactivity pattern in molecular layer and in glomerula of granule cell layer of the cerebellum in patient 7 (A1), in a comparable pattern and intensity as can be observed in a patient with Alzheimer’s disease (AD) used as control (B1). There is only very faint cytoplasmic staining of Purkinje cells. A2, B2: In a section through medulla oblongata, the most affected area in patient 7 (A1), the staining pattern is again comparable to the patient with Alzheimer’s disease without brainstem involvement (B2), and shows distinct staining of neuronal processes and a faint immunoreactivity of perikarya.

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