Diagnosis of a neurological disease

Abstract

The present invention concerns subject matter connected to or making use of IgLON5, IgLON5 fragments and variants of IgLON5 and IgLON5-fragments. In particular the present invention relates to a use of a polypeptide comprising one or more sequences of IgLON5, an IgLON5-fragment or a variant thereof for the diagnosis of a disease, in vitro methods for diagnosing such a disease, a polypeptide comprising one or more sequences of IgLON5, an IgLON5-fragment or a variant thereof or a nucleic acid encoding said polypeptide for use in the treatment of a disease, a pharmaceutical composition comprising such polypeptide, a method for treating such a disease, an autoantibody binding to IgLON5, an IgLON5-fragment or a variant thereof, a method for isolating such autoantibody, a medical or diagnostic device comprising such autoantibody or such polypeptide and a test kit for the diagnosis of a disease, which test kit comprises such autoantibody and/or such polypeptide.

Claims

1. A method of detecting an autoantibody to IgLON5 or a fragment thereof in a subject, the method comprising: contacting a bodily fluid sample isolated from a subject with a polypeptide comprising IgLON5 or an IgLON5 fragment; and detecting the presence or absence of an autoantibody to IgLON5 in a complex with the polypeptide, wherein the IgLON5 comprises an amino acid sequence SEQ ID NO:1 or SEQ ID NO:12, and wherein the IgLON5 fragment comprises an amino acid sequence selected from a group of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5; SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, and SEQ ID NO:11.

2. The method of claim 1, wherein the polypeptide is immobilized on a solid carrier.

3. The method of claim 1, wherein the detecting the presence or absence of the autoantibody to IgLON5 in a complex with the polypeptide comprises the use of a tagged or labeled secondary antibody.

4. The method of claim 1, wherein the polypeptide is provided in the form of a cell comprising a nucleic acid encoding the polypeptide or in the form of a tissue comprising the polypeptide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows a sleep recording in patient. A. Hypnogram; B. Arousals, dissociations and periodic movements; C. Density Spectral Array (DSA) showing the power spectrum of electroencephalographic frequencies (0-17 Hz) in electrode C3 referenced to electrode O2. Abbreviations: Rems Rapid eye movements; RBD: REM sleep behavior disorder; PLM: Periodic limb movements; RPLM: Rapid periodic leg movements.

(2) FIG. 2 shows polysomnograhic epochs illustrative of each sleep state, see also FIG. 1. A: Sleep onset characterized by undifferentiated NREM sleep with diffuse theta activity and rapid periodic leg movements that were particularly prominent at the left AT EMG channel; B: N2 sleep with K complexes in a chain of quadruplet (arrows), with frequent aperiodic muscular phasic activity in EMG surface of the limbs that correlate with gesticulations and vocalizations; C: REM sleep with typical rapid eye movements and EEG features with excessive phasic and tonic muscular activity and body jerks typical of REM sleep behaviors disorder; D: N3 with diffuse delta activity and well defined sleep spindles at 13 Hz (arrows) without body/limb movements. Abbreviations: EEG: electroencephalogram EMG; electromyogram; EOG: electrooculogram; Chin: Electromyography of mentalis muscle; EKG: Electrocardiogram; FDS: Flexor digitorum superficialis muscle left (L) and right (R); EDB: Extensor digitorum brevis muscle left (L) and right (R); AT: Anterior tibialis left (L) and right (R); NAS: Nasal air flow; THO: Thoracic respiratory movement; ABD; Abdominal respiratory movement; Note the calibration mark for time/EEG voltage.

(3) FIG. 3 shows the reactivity of patient's antibodies with rat brain and cultures of hippocampal neurons. (A) Sagittal section of rat brain immunostained with a patient's CSF: there is a diffuse staining in the neuropil not seen when rat brain sections are incubated with a control CSF (B). The immunoreactivity was particularly robust in the cerebellum (C) where there was diffuse staining of the molecular layer and synaptic glomerula of the granular cell layer (D). (E) Culture of rat hippocampal neurons incubated (nonpermeabilized) with a patient's serum showing intense reactivity with a cell surface antigen. (D) Counterstained with hematoxylin. Scale bars in A and B=1000 m, C=200 m, D=50 m and E=20 m.

(4) FIG. 4 shows the IgG isotype analysis of antibodies against IgLON5. Reactivity of a patient's serum with rat hippocampus after incubation with antibodies specific for human IgGI (A), IgG2 (B), IgG3 (C), and IgG4 (D). Robust neuropil immunostaining is only observed with IgG4. Scale bar=200 m.

(5) FIG. 5 shows the detection of IgLON5 antibodies using a HEK293 cell based assay. HEK293 cells were transfected to express EGFP-tagged IgLON5 and incubated live, not permeabilized, with a patient's (A-C) or control (D-F) serum. Patient's serum, but not control serum, stained the cell surface of cells (lighter grey) that specifically express IgLON5, as demonstrated by the EGFP fluorescence (lighter grey). Both reactivities are shown merged in C. Nuclei counterstained with DAPI. Scale bar=20 m.

(6) FIG. 6 shows the distribution of tau pathology. Panels A1-F1 correspond to patient 2, and panels A2-F2 correspond to patient 5. Moderate amounts of AT8 immunoreactive neuropil threads and neurofibrillary tangles are detected in hypothalamic nuclei (B1, A2. posterior hypothalamic nucleus; example of score ++) and anterior thalamus (A1, B2: left figure), but are completely absent in lateral and posterior thalamic neurons of both cases (A1, B2, right figure; example of score 0). While the pontine tegmentum is mildy (D2; example of score +) and moderately (C1) affected in case 5 and case 2, respectively, neurons of n. propii of basis pontis show extensive Tau-pathology (D1; example of score +++), which is not observed in case 5 (C2). In contrast, prominent pathology in n. ambiguus is detected in case 5 (F2; example of score +++), and less in case 2 (E1) and to a lesser extent in magnocellular nuclei of formatio reticularis in both cases (F1, E2).

(7) FIG. 7 shows 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).

(8) FIG. 8 shows 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 m, C and D=20 m.

(9) Throughout this application, a number of sequences are disclosed which are referred to as:

(10) SEQ ID NO 1: human IgLON5

(11) SEQ ID NO 2: rodent/human IgLON5 fragment, see FIG. 7

(12) SEQ ID NO 3: human IgLON5 fragment

(13) SEQ ID NO 4: rodent/human IgLON5 fragment, see FIG. 7

(14) SEQ ID NO 5: rodent/human IgLON5 fragment, see FIG. 7

(15) SEQ ID NO 6: rodent/human IgLON5 fragment, see FIG. 7

(16) SEQ ID NO 7: rodent/human IgLON5 fragment, see FIG. 7

(17) SEQ ID NO 8: rodent/human IgLON5 fragment, see FIG. 7

(18) SEQ ID NO 9: rodent/human IgLON5 fragment, see FIG. 7

(19) SEQ ID NO 10: rodent/human IgLON5 fragment, see FIG. 7

(20) SEQ ID NO 11: rodent IgLON5 fragment, see FIG. 7

(21) SEQ ID NO 12: artificial IgLON5 construct, see FIG. 7

REFERENCES

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EXAMPLES

(23) Patients and Methods

(24) Inclusion Criteria, Patients, and Controls

(25) Three of the eight patients (patients 1-3, Table 1) of this study were from the cohort of patients studied in the multidisciplinary sleep disorders unit of Hospital Clinic, Barcelona, Spain. The remaining five patients were identified among samples sent to our laboratory with similar immunohistochemical reactivity. Serum or CSF of 251 patients were used as controls including 45 with pathologically confirmed Alzheimer disease, 28 with clinical diagnosis of progressive supranuclear palsy, 21 with DNA-binding protein 43 (TDP-43) frontotemporal dementia, 18 with multiple system atrophy, 25 with idiopathic RBD, 28 with hypocretin deficient narcolepsy, 54 with multiple sclerosis, and 32 with anti-Lgi1 encephalitis. Brain tissue, serum and CSF samples used in the study are deposited in the Neurological Tissue Bank and the Biobank of the Institut d'Investigacions Biomdiques August Pi i Sunyer, Barcelona, Spain.

(26) Polysomnographic (PSG) Studies

(27) Nocturnal video-polysomnography included electrooculography, electroencephalography (F3, F4, C3, C4, O1 and O2, referred to combined ears), submental EMG, surface EMG of the right and left anterior tibialis, flexor digitorum superficialis in the upper limbs, and the extensor digitorum brevis in the lower limbs. Electrocardiography, nasal and oral airflow, thoracic and abdominal movements, and oxyhemoglobin saturation were also recorded.

(28) We scored undifferentiated NREM sleep epochs of irregular theta EEG slowing, clearly different from the awake alpha rhythm, and lacking vertex sharp waves, K complexes, sleep spindles or delta slowing, and without definite and recurrent rapid eye movements, such as those typically seen in later periods of REM sleep. We scored stage N2 epochs with definite K complexes or spindles at 12-14 Hz, even if associated with excessive EMG activation and movements or occasional rapid eye movements of low amplitude than those typical of the REM sleep in the same patient. Encapsulated rapid eye movement sleep behavior disorder (RBD) during NREM sleep occurred when an episode lasting few seconds contained rapid eye movements, excessive EMG twitching and typical RBD jerks together with EEG features of REM sleep within an epoch of NREM sleep. Vocalizations were scored as simple and complex in each epoch, and body/limb movements as jerks, simple or finalistic depending upon their video characteristics (jerks were sudden contractions of a single or several muscle groups; finalistic were movements following a pattern that clearly reminded an identifiable daytime activitye.g. eating, drinking, manipulating objects, etc.; and simple were the movements that were more complex than jerks but no so elaborated as the finalistic ones). We defined rapid periodic leg movements as those periodic leg movements that occurred with an interval between movements shorter than 5 seconds.

(29) Four patients were recorded with video-PSG several nights throughout their clinical course at the sleep laboratory of the Hospital Clinic of Barcelona (Patients 1-3 and 5), and one patient at the sleep laboratory in Ulm, Germany (Patient 7). In total nineteen video-PSG were recorded. EMG was recorded in the submentalis muscle and both upper and lower limbs. Sleep stages and associated events were scored according to the 2007 American Academy of Sleep Medicine criteria (Iber C., Ancoli-Israel S., Chesson A., and Quan S. F. (2007), The AASM manual for the scoring of sleep and associated events: rules, terminology and technical specifications, 1st ed. Westchester, Ill.: American Academy of Sleep Medicine) or with the modifications previously proposed when the standard criteria could not be followed (Santamaria J., Hgl B., Trenkwalder C., Bliwise D. (2011), Scoring sleep in neurological patients: the need for specific considerations, Sleep 2011; 34:1283-4).

(30) Procedures for Detection of IgLON5 Antibodies and Characterization of the Antigen

(31) 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 (Lancaster E., Dalmau J. (2012), Neuronal autoantigenspathogenesis, associated disorders and antibody testing, Nat. Rev. Neurol. 2012; 8:380-90). 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, Ala., USA) as described (Cornelius J. R., Pittock S. J., McKeon A., et al. (2011), Sleep manifestations of voltage-gated potassium channel complex autoimmunity, Arch. Neurol. 2011; 68:733-8).

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

(33) Rat hippocampal neuronal cultures were prepared as reported (Iranzo A., Graus F., Clover L., et al. (2006), Rapid eye movement sleep behavior disorder and potassium channel antibody-associated limbic encephalitis, Ann Neurol 2006; 59:178-81). Fourteen days live neurons grown on coverslips were treated for 1 hour at 37 C. with patients' or control serum (final dilution 1:750) or CSF (1:30). 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, N.Y.) (Montagna P., Lugaresi E. (2002), Agrypnia Excitata: a generalized overactivity syndrome and a useful concept in the neurophysiopathology of sleep, ClinNeurophysiol 2002; 113:552-60).

(34) Immunoprecipitation experiments were done with cultures of rat hippocampal neurons grown in 100 mm 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 for 20 minutes at 4 C. The supernatant was retained, incubated with protein A/G agarose beads (20423; Pierce, Rockford, Ill.) 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, Calif.) as reported (Irani S. R., Pettingill P., Kleopa K. A., et al. (2012), Morvan syndrome: clinical and serological observations in 29 cases, Ann Neurol 2012; 72:241-55). The Xcalibur software (Thermo Scientific, Waltham, Mass.) was utilized to acquire the raw data and Sequest program (ThermoFinnigan, San Jose, Calif.; 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.

(35) To further confirm the specificity of the antigen, HEK293 cells were transfected with plasmids containing IgLON1, 2, 3, 4, and 5 (GFP-tagged clones from Origene: RG213594, RG226879, RG207618, RG216034, RG225495) as described (Dalmau J., Gleichman A. J., Hughes E. G., et al. (2008), Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies, Lancet Neurol. 2008; 7:1091-8). Cells were grown for 24 hours after transfection, incubated for 1 hour at 37 C. with patients' or control serum (final dilution 1:40) or CSF (1:2), and fixed with 4% paraformaldehyde, and permeabilized with 0.2% Triton X-100 (Sigma, Saint Louis, Mo.). Immunolabeling was performed using the appropriate Alexa-Fluor secondary antibodies diluted 1:1000 (Molecular Probes, OR) (Dalmau J., Gleichman A. J., Hughes E. G., et al. (2008), Anti-NMDA-receptor encephalitis: case series and analysis of the effects of antibodies, Lancet Neurol. 2008; 7:1091-8).

(36) To rule out the possibility of additional neuronal antibodies, serum diluted 1:200 was serially incubated with six wells containing live HEK293 cells expressing IgLON5 or cells transfected with plasmids without insert. After sequential passes of one hour each, the serum was applied to sections of rat brain and live hippocampal neurons and the reactivity developed using the methods described above.

(37) Neuropathological Studies

(38) Neuropathologic examination was performed according to standardized protocols at the Neurological Tissue Bank of the IDIBAPS Biobank in two patients (patients 2 and 5 Table 1) (Gelpi E., Llad A., Clarimn J., et al. (2012), Phenotypic variability within the inclusion body spectrum of basophilic inclusion body disease and neuronal intermediate filament inclusion disease in frontotemporal lobar degenerations with FUS-positive inclusions, J Neuropathol Exp Neurol 2012; 71:795-805). Immunohistochemistry was performed applying a panel of primary antibodies (Table 1S) as described (Gelpi E., Llad A., Clarimn J., et al. (2012), Phenotypic variability within the inclusion body spectrum of basophilic inclusion body disease and neuronal intermediate filament inclusion disease in frontotemporal lobar degenerations with FUS-positive inclusions, J Neuropathol Exp Neurol 2012; 71:795-805). To evaluate the expression of IgLON5 in the areas more affected by tau pathology, sections from one of the autopsy cases and similar areas from a patient with Alzheimer disease were incubated, with a commercial antibody against IgLON5 (Abcam, Cambridge, UK) and the immunoreactivity visualized with the avidin-biotin immunoperoxidase method.

(39) Results

(40) Clinical Findings

(41) The eight patients (5 women; age range: 52 to 76 years) had a prominent sleep disorder characterized by abnormal sleep movements and behaviors and obstructive sleep apneas with stridor. Five patients (patients 1-4 and 8, Table 1) were initially diagnosed of isolated obstructive sleep apnea syndrome. However, continuous positive airway pressure (CPAP) therapy improved the stridor and obstructive sleep apnea but not the other sleep symptoms. In four patients (Patients 1 to 4, Table 1), the sleep disorder was the presenting and most prominent complaint during the entire course of the disease. In addition to the sleep disorder, two patients (Patients 5 and 6) developed severe gait difficulties with loss of balance and gait failure, dysarthria, dysphagia, vocal cord paralysis, and central hypoventilation that was the cause of death in less than 6 months. The last two patients (Patients 7 and 8) had a chronic progressive evolution that started with frequent falls and gait instability followed by dysarthria, dysphagia, limb ataxia, and choreic movements in the limbs and face. In addition most patients developed mild memory and attention deficits with apathy, depressed mood, akathisia, or urinary disturbances. None of the patients developed parkinsonism or oculomotor signs compatible with the diagnosis of progressive supranuclear palsy. The clinical features and outcome are described in detail in Table 1.

(42) Brain MRI, routine electroencephalogram, and CSF analysis were unremarkable. Nerve conduction studies and EMG done in seven patients ruled out neuromyotonia. CSF hypocretin levels obtained in three patients were normal. Human leukocyte antigen (HLA) typing was performed in four patients and all showed the HLA-DQB1*0501 and HLA-DRB1*1001 alleles. Three of them (patients 1-3) were also HLA-B27 positive. All patients received some type of immunotherapy without substantial improvement. Six patients are dead and all had sudden death or presented severe central hypoventilation (Table 1).

(43) Polysomnographic Studies

(44) The most prominent features of PSG studies are summarized in FIG. 1 and Table 2. Total sleep time was slightly reduced (sleep efficiency 68-88%; normal >85%). In four patients, sleep onset was accompanied by rapid periodic leg movements (FIG. 2A, video segment 1). Initiation of sleep, as well as reentering sleep after midnight awakenings, was abnormal in all five patients, either as an undifferentiated NREM sleep, in four patients, or poorly structured N2 sleep in one. There were frequent vocalizations, simple non-purposeful or finalistic movements with a pattern resembling daytime activities such as eating, drinking or manipulating objects (FIG. 2B, video segments 2-5). Normal N1 sleep was absent and well-structured N2 sleep with K complexes and spindles was rare. REM sleep was recorded in 16 out of the 19 PSG recordings and always in the form of RBD of mild to moderate severity (FIG. 2C). In all patients, clear periods of delta slowing typical of N3 sleep with frequent spindles and without vocalizations or movements, were recorded. Finally, all patients presented a sleep breathing disorder with stridor and moderate-severe obstructive sleep apneas (apnea-hypoapnea index without CPAP ranging from 20 to 84 apneas-hypoapneas per hour; normal 5) that were worse during quiet N3 sleep.

(45) There was a characteristic distribution throughout the night of these sleep abnormalities (present in 14 out of the 19 PSG recordings). Periods of undifferentiated NREM and poorly structured N2 with finalistic movements predominated and were of longer duration after onset of nocturnal sleep or following awakenings in the first half of the night. Normal N3 sleep and RBD were more frequent and lasted longer in the second half of the night (FIG. 2D).

(46) Antibody Characterization

(47) The serum of the eight patients, and the five CSF available, showed an identical pattern of reactivity with the neuropil of rat brain. The immunoreactivity was more intense in the molecular layer and synaptic buttons of the granular layer of the cerebellum (FIG. 3). All sera and CSF labeled the membrane of live neurons in culture indicating the antigen was exposed on the cell surface (FIG. 3). Immunocompetition assays showed that all samples blocked the reactivity of the biotinylated IgG obtained from the serum of Patient 1 strongly suggesting that the antibodies of the eight patients reacted with the same epitopes.

(48) Initial serum antibody titers ranged from 1/5000 to 1/40000. In four patients, the titers decreased more than two-fold in the follow-up samples obtained after immunotherapy (Table 1). By contrast, no change in antibody titers was observed in the follow-up of another patient during the year that he was not treated. Analysis of IgG subclasses showed that in all patients the novel neuropil-reacting antibody was IgG4 (FIG. 4); one patient had additional IgG2, and four had very mild IgG1 reactivity. None of the patients had IgM antibody reactivity.

(49) Identification of IgLON Family Member 5 as the Targeted Antigen

(50) Mass spectrometry analysis revealed IgLON5 in two independent immunoprecipitation experiments using the serum of two patients. In every experiment we included as negative control a normal human serum. Seven peptides containing 29% of the protein sequence, and 3 peptides containing 14% of the protein sequence of IGLON5 were isolated using the antibodies of the two patients (FIG. 7). To further confirm the specificity of patients' antibodies for IgLON5, HEK293 cells transfected with plasmids coding each of the five members of the IgLON family were used in a cell-based assay (CBA). The serum and CSF of the eight patients only reacted with cells transfected with the GFP-tagged IgLON5 (FIG. 5). Sera did not react with HEK cells transfected with GFP-tagged IgLON1, 2, 3 or 4, thus confirming the antibody reactivity was specific for IgLON5 and not directed to the GFP tag (not shown). Analysis of serum or CSF of the 251 controls using the IgLON5 CBA identified a patient with antibodies in serum, but not CSF, who had been clinically diagnosed with progressive supranuclear palsy. The rest of the controls were negative.

(51) To determine if patients' serum contained additional antibodies that could explain the additional symptoms of some cases (e.g., ataxia, hypoventilation), we immunoabsorbed the serum of three patients with different clinical course (Patients 1, 5, 7) with HEK293 cells expressing IgLON5 or cells transfected with plasmids without insert. The absorption with IgLON5 completely abrogated the reactivity of the three sera with rat brain and hippocampal neurons indicating that patients' antibodies were directed only against IgLON5 (FIG. 8).

(52) Neuropathological Examination

(53) The autopsy of the two patients showed a neuronal tauopathy with predominant involvement of hypothalamus, prehypothalamic region, and tegmentum of brainstem including laterodorsal tegmental area, periaqueductal gray matter, the region of the pedunculopontine nucleus, magnocellular nuclei, and nucleus ambiguus (Table 3, FIG. 6). The Tau aggregates were exclusively neuronal, in form of pretangles, tangles and neuropil threads, with presence of 3-repeat and 4-repeat tau isoforms in patient 5 and predominance of 4-repeat tau isoforms in patient 2. Neurofibrillary pathology showed strong immunoreactivity for phospho-specific anti-tau antibodies Thr181, Ser262, Ser396, Ser422 (not shown). There were no tau-positive grains and no glial tau pathology, neither in astrocytes (tufted or thorn shaped astrocytes, bush-like or peculiar astrocytes or astrocytic plaques), nor in oligodendrocytes (coiled bodies, globular glial inclusions). No inflammatory infiltrates or concomitant abnormal protein deposits of beta-amyloid, alpha-synuclein, and TDP43 were detected. IgLON5 immunoreactivity was not reduced in the affected brainstem regions of patient 5 compared with those of a patient with Alzheimer disease used as control.

(54) TABLE-US-00001 TABLE 1 Clinical features, treatment and outcome. Patient Presenting Disease Cognitive and Sex/age symptoms duration Bulbar psychiatric Treatment at onset (onset) (course) Sleep problems Gait Symptoms problems Other symptoms and outcome Patient 1 Sleep 4 years Sleep movements and Normal Right VC Mild memory and Akathisia. 3 cycles of iv M/59 problems (insidious) behaviors.* Stridor with paresis attention Hypersalivation. steroids Ig, Cy (chronic) OSA. Fragmented sleep. complaints. Episodes of No change Occasional confusional Apathy, low intense awakenings.** Intermittent mood perspiration. intense EDS Patient 2 Sleep 6 years Sleep movements and Normal Mild Mild memory and Akathisia 3 cycles of iv M/53 problems (insidious/ behaviors.* Stridor with dysphagia attention steroids Ig, Cy (chronic) fluctuating) OSA. Fragmented sleep. complaints. No change Occasional confusional Apathy, low Sudden death awakenings.** Nocturnal mood while asleep enuresis. Mild EDS Patient 3 Sleep 5 years Sleep movements and Mild Mild Mild memory and Akathisia 3 cycles of iv M/52 problems (insidious/ behaviors.* Stridor with unsteadiness dysphagia attention Chorea steroids Ig, Cy (subacute) fluctuating) OSA. Intense episodic EDS infrequent and complaints. No change falls dysarthria. Apathy, low Bilateral VC mood paresis Patient 4 Sleep 2 years Sleep movements. Loud No Dysarthria. Memory Chorea. Syncopes Iv steroids. F/69 problems (progressive) snoring with OSA. Episodes of complaints with hypotension No change (subacute) Fragmented steep. central Anxiety and bradycardia. Sudden death Nocturnal confusional hypo- Mild vertical gaze awakenings. ventilation palsy Moderate EDS Patient 5 Gait 6 months Sleep movements and Gait failure Dysphagia. Depressed mood Mild oculomotor Iv/oral F/76 instability (rapidly behaviors* with severe Bilateral VC dysfunction with steroids with falls progressive) Stridor with OSA postural paresis. saccadic No change (subacute) instability Central intrusions on Sudden death hypo- pursuit while asleep ventilation Patient 6 Gait 2 months Frequent sleep movements Gait failure Dysphagia. Long history of Bilateral Iv steroids, Ig, F/65 difficulties (rapidly and behaviors. Loud with severe Mandibular severe chronic horizontal and rituximab (subacute) progressive) snoring with OSA. postural spasms. depression nystagmus. Improved.*** Fragmented sleep instability Central Limba taxia. Sudden death hypo- during ventilation daytime Patient 7 Gait 12 years Frequent sleep movements Cerebellar Dysphagia Mental slowness Chorea Iv/oral F/59 instability (slowly and behaviors. Loud ataxia. Dysarthria with memory and Bradykinesia steroids; with falls progressive) snoring. Mild EDS Severe attention Limb ataxia rituximab (chronic) postural difficulties No change instability Depressed mood Sudden death during daytime Patient 8 Gait 5 years Frequent sleep movements Cerebellar Dysphagia Mild memory and Chorea Iv Ig F/58 instability (slowly and behaviors. Stridor with ataxia Dysarthria attention Vertical/ No change with falls progressive) OSA. Fragmented sleep. Central complaints horizontal Dead from (chronic) hypo- Depressed mood nystagmus with central ventilation saccadic hypoventilation intrusions Limb dysmetria Cy: cyclophosphamide; EDS: Excessive daytime sleepiness; Ig: immunoglobulins; OSA: sleep obstructive apnea; VC: Vocal cord. *Video-polysomnography demonstrated a NREM and REM sleep parasomnia as the underlying substrate for the abnormal sleep movements and behaviors. **Confusional awakening related to the anticholinergic effect of tricyclic antidepressants. ***Patient initially improved and could be discharged but she made a sudden death 2 days later

(55) TABLE-US-00002 TABLE 2 Polysomnographic characteristics 1. Total sleep time is moderately reduced. 2. A distinctive temporal sequence of sleep stages and behaviours occurs, from most abnormal at the beginning of the night, to more normal at the end. 3. Rapid periodic leg movements during wakefulnes are often present, and they continue after sleep onset. 4. Initiation of sleep and re-entering sleep after awakening is abnormal with undifferentiated NREM sleep or poorly structured N2 sleep stage with frequent vocalizations, stereotyped repetitive upper limb movements and/or finalistic behaviors. 5. Normal N1 sleep stage is absent. Normal well-structured N2 sleep stage is infrequent or absent. Diffuse delta activity, typical of normal N3 sleep stage is present and always associated with frequent spindles. 6. REM sleep is present but only in the form of RBD 7. Sleep breathing disorder characterized by stridor and obstructive sleep apneas RBD: REM sleep behavior disorder

(56) TABLE-US-00003 TABLE 3 Topographical distribution of neuronal loss and tau pathology Patient 2 (table1) Patient 5 (table 1) neuronal loss tau neuronal loss tau Brain region 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 + ++ + +++ Periaquaeductal grey ++ + + ++ Substantia nigra 0 0* 0 0* Locus coeruleus 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 200 or 400 magnification.