U.S. patent application number 15/116179 was filed with the patent office on 2016-12-01 for diagnosis of a neurological disease.
This patent application is currently assigned to INSTITUT D'INVESTIGACIONES BIOM DIQUES AUGUST PII SUNYER. The applicant listed for this patent is HOSPITAL CL NIC DE BARCELONA, INSTITUCIO CATALANA DE RECERCA I ESTUDIS AVAN ATS, INSTITUT D'INVESTIGACIONES BIOM DIQUES AUGUST PI I SUNYER, UNIVERSITAT DE BARCELONA. Invention is credited to Josep DALMAU, Francesc GRAUS.
Application Number | 20160349275 15/116179 |
Document ID | / |
Family ID | 50068912 |
Filed Date | 2016-12-01 |
United States Patent
Application |
20160349275 |
Kind Code |
A1 |
DALMAU; Josep ; et
al. |
December 1, 2016 |
DIAGNOSIS OF A NEUROLOGICAL DISEASE
Abstract
The present invention concerns subject matter connected to or
making use of lgLON5, lgLON5 fragments and variants of lgLON5 and
lgLON5-fragments. In particular the present invention relates to a
use of a polypeptide comprising one or more sequences of IgLON5, an
lgLON5-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 lgLON5, an
lgLON5-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
lgLON5-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.
Inventors: |
DALMAU; Josep; (Barcelona,
ES) ; GRAUS; Francesc; (Barcelona, ES) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
INSTITUT D'INVESTIGACIONES BIOM DIQUES AUGUST PI I SUNYER
INSTITUCIO CATALANA DE RECERCA I ESTUDIS AVAN ATS
UNIVERSITAT DE BARCELONA
HOSPITAL CL NIC DE BARCELONA |
Barcelona
Barcelona
Barcelona
Barcelona |
|
ES
ES
ES
ES |
|
|
Assignee: |
INSTITUT D'INVESTIGACIONES BIOM
DIQUES AUGUST PII SUNYER
Barcelona
ES
INSTITUCIO CATALANA DE RECERCA I ESTUDIS AVAN ATS
Barcelona
ES
UNIVERSITAT DE BARCELONA
Barcelona
ES
HOSPITAL CL NIC DE BARCELONA
Barcelona
ES
|
Family ID: |
50068912 |
Appl. No.: |
15/116179 |
Filed: |
February 6, 2015 |
PCT Filed: |
February 6, 2015 |
PCT NO: |
PCT/EP2015/052529 |
371 Date: |
August 2, 2016 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 2800/2814 20130101;
G01N 33/6854 20130101; G01N 2800/2864 20130101; G01N 2800/2821
20130101; G01N 33/564 20130101; C07K 16/2803 20130101; G01N 33/6896
20130101 |
International
Class: |
G01N 33/68 20060101
G01N033/68; C07K 16/28 20060101 C07K016/28; G01N 33/564 20060101
G01N033/564 |
Foreign Application Data
Date |
Code |
Application Number |
Feb 7, 2014 |
EP |
14154384.3 |
Claims
1. (canceled)
2. A polypeptide comprising one or more sequences of IgLON5, an
IgLON5-fragment, or a variant thereof or a nucleic acid encoding
said polypeptide.
3. An autoantibody binding to at least one of the polypeptides of
claim 2.
4-16. (canceled)
17. The polypeptide of claim 2, wherein the sequence of IgLON5 is
the sequence according to SEQ ID NO:1, and wherein the sequence of
the IgLON5-fragment is a sequence selected from the group
consisting 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, and SEQ ID
NO:10.
18. The autoantibody of claim 3, 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.
19. An in vitro method for diagnosing a disease, comprising the
steps of: a1) contacting a liquid sample comprising an antibody
from a subject with a polypeptide of claim 2, or a2) contacting a
tissue sample comprising IgLON5, an IgLON5-fragment, or a variant
thereof from a subject with an antibody binding to IgLON5, the
IgLON5-fragment, or the variant thereof, and, following steps a1)
or a2), b) detecting formation of a complex comprising the antibody
and IgLON5, the IgLON5-fragment, or the variant thereof.
20. The in vitro method of claim 19, comprising step a2), wherein
the antibody is an autoantibody binding to IgLON5, the
IgLON5-fragment, or the variant thereof.
21. The in vitro method of claim 19, wherein the disease is
selected from the group consisting of sleep disorders,
neurodegenerative diseases, and hypoventilation.
22. The in vitro method of claim 19, wherein the disease is
associated with one or more symptoms selected from the group
consisting of parasomnia, stridor, sleep apnea, gait instability,
dysarthria, dysphagia, limb ataxia, vocal cord paralysis, choreic
movements in the limbs and face, memory and attention deficits,
apathy, depressed mood, akathisia, and urinary disturbances.
23. The in vitro method of claim 19, wherein the sequence of IgLON5
is the sequence according to SEQ ID NO:1, and wherein the sequence
of the IgLON5-fragment is a sequence selected from the group
consisting 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, and SEQ ID
NO:10.
24. The in vitro method of claim 19, comprising step a1), wherein
the polypeptide comprising one or more sequences of IgLON5, the
IgLON5-fragment, or the variant thereof 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.
25. A test kit for the diagnosis of a disease, comprising at least
one of: an autoantibody binding to IgLON5, an IgLON5-fragment, or a
variant thereof; or a polypeptide comprising one or more sequences
of IgLON5, an IgLON5-fragment, or a variant thereof.
26. The test kit of claim 25, comprising the polypeptide comprising
one or more sequences of IgLON5, the IgLON5-fragment, or the
variant thereof, wherein the polypeptide is provided in an
immobilized form.
27. The test kit of claim 25, further comprising a means for
detecting a complex comprising an antibody and IgLON5, the
IgLON5-fragment, or the variant thereof.
28. The test kit of claim 25, wherein the disease is selected from
the group consisting of sleep disorders, neurodegenerative
diseases, and hypoventilation.
29. The test kit of claim 25, wherein the disease is associated
with one or more symptoms selected from the group consisting of
parasomnia, stridor, sleep apnea, gait instability, dysarthria,
dysphagia, limb ataxia, vocal cord paralysis, choreic movements in
the limbs and face, memory and attention deficits, apathy,
depressed mood, akathisia, and urinary disturbances.
30. The test kit of claim 25, wherein the sequence of IgLON5 is the
sequence according to SEQ ID NO:1, and wherein the sequence of the
IgLON5-fragment is a sequence selected from the group consisting 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, and SEQ ID NO:10.
31. The test kit of claim 25, comprising the polypeptide comprising
one or more sequences of IgLON5, the IgLON5-fragment, or the
variant thereof, 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.
32. A method for isolating an autoantibody binding to IgLON5, an
IgLON5-fragment, or a variant thereof, the method comprising the
steps of: a) contacting a sample comprising the autoantibody with a
polypeptide of claim 2, such that a complex is formed; b) isolating
the complex formed in step a); c) dissociating the complex isolated
in step b); and d) separating the autoantibody from the polypeptide
comprising one or more sequences of IgLON5, the IgLON5-fragment, or
the variant thereof.
33. The method of claim 32, wherein the polypeptide comprising one
or more sequences of IgLON5, the IgLON5-fragment, or the variant
thereof is immobilized.
34. The method of claim 32, wherein the sequence of IgLON5 is the
sequence according to SEQ ID NO:1, and wherein the sequence of the
IgLON5-fragment is a sequence selected from the group consisting 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, and SEQ ID NO:10.
35. The method of claim 32, wherein the polypeptide comprising one
or more sequences of IgLON5, the IgLON5-fragment, or the variant
thereof 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.
36. A method for treating a disease associated with one or more
symptoms selected from the group consisting of parasomnia, stridor,
sleep apnea, gait instability, dysarthria, dysphagia, limb ataxia,
vocal cord paralysis, choreic movements in the limbs and face,
memory and attention deficits, apathy, depressed mood, akathisia,
and urinary disturbances in a subject, comprising the steps of: a)
reducing the concentration of autoantibodies binding to IgLON5, an
IgLON5-fragment, or a variant thereof in the subject's blood;
and/or b) administering one or more immunosuppressive
pharmaceutical substances to the subject.
37. The method of claim 36, comprising administering the one or
more immunosuppressive pharmaceutical substances to the subject,
wherein the immunosuppressive pharmaceutical substances are
selected from the group consisting of rituximab, prednisone,
methylprednisolone, cyclophosphamide, mycophenolatemofetil,
intravenous immunoglobulin, tacrolimus, cyclosporine, methotrexate,
azathioprine, and/or a polypeptide comprising one or more sequences
of IgLON5, an IgLON5-fragment, or a variant thereof.
38. The method of claim 36, wherein the sequence of IgLON5 is the
sequence according to SEQ ID NO:1, and wherein the sequence of the
IgLON5-fragment is a sequence selected from the group consisting 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, and SEQ ID NO:10.
39. A method of detecting the presence or absence of autoantibodies
in a subject, comprising: obtaining a sample from a subject;
contacting the sample with a polypeptide comprising one or more
sequences of IgLON5, an IgLON5-fragment, or a variant thereof; and
detecting in the sample the presence or absence of at least one
complex comprising the polypeptide and an autoantibody binding to
the polypeptide.
40. The method of claim 39, wherein the polypeptide is tagged,
labeled, or immobilized.
41. A method of diagnosing and/or treating a disease in a subject,
the method comprising: obtaining a sample from a subject;
contacting the sample with a polypeptide comprising one or more
sequences of IgLON5, an IgLON5-fragment, or a variant thereof;
detecting in the sample the presence or absence of at least one
complex comprising the polypeptide and an autoantibody binding to
the polypeptide; and diagnosing the subject with a disease selected
from the group consisting of sleep disorders, neurodegenerative
diseases, and hypoventilation when the presence of the at least one
complex is detected.
42. The method of claim 41, further comprising at least one of (i)
administering one or more immunosuppressive pharmaceutical
substances to the subject, and (ii) reducing the concentration of
autoantibodies binding to IgLON5, an IgLON5-fragment, or a variant
thereof in the subject.
Description
[0001] The present application claims priority of European Patent
application EP 1 415 4381, the contents of which are incorporated
in the present application in their entirety.
[0002] 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, 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, an
autoantibody binding to IgLON5, an IgLON5-fragment or a variant
thereof, which is preferably for use in the diagnosis of a disease,
a pharmaceutical composition comprising a polypeptide comprising
one or more sequences of IgLON5, an IgLON5-fragment or a variant
thereof, an in vitro method for diagnosing a disease, comprising
the steps a1) contacting a liquid sample comprising an antibody
from a subject with a polypeptide comprising one or more sequences
of IgLON5, an IgLON5-fragment or a variant thereof, or a2)
contacting a tissue sample comprising IgLON5, an IgLON5-fragment or
a variant thereof from a subject with an antibody binding to
IgLON5, the IgLON5-fragment or the variant thereof, and following
steps a1) or a2), b) detecting formation of a complex comprising
the antibody and IgLON5, the IgLON5-fragment or the variant
thereof, an in vitro method for diagnosing a disease, preferably an
autoimmune disease, comprising the step of detecting in a liquid
sample comprising antibodies an autoantibody binding to IgLON5, a
medical or diagnostic device comprising such autoantibody or such
polypeptide, a test kit for the diagnosis of a disease, which test
kit comprises the autoantibody and/or a polypeptide comprising one
or more sequences of IgLON5, an IgLON5-fragment or a variant
thereof, a method for isolating an autoantibody binding to IgLON5,
an IgLON5-fragment or a variant thereof, comprising the steps a)
contacting a sample comprising the autoantibody with a polypeptide
comprising one or more sequences of IgLON5, the IgLON5-fragment or
the variant thereof, which polypeptide is preferably immobilized,
such that a complex is formed, b) isolating the complex formed in
step a), c) dissociating the complex isolated in step b), and d)
separating the autoantibody from the polypeptide comprising one or
more sequences of IgLON5, the IgLON5-fragment or the variant
thereof, and a method for treating a disease associated with one or
more symptoms selected from the group comprising parasomnia,
stridor, sleep apnea, gait instability, dysarthria, dysphagia, limb
ataxia, vocal cord paralysis, choreic movements in the limbs and
face, memory and attention deficits, apathy, depressed mood,
akathisia and urinary disturbances in a subject, comprising the
steps a) reducing the concentration of autoantibodies binding to
IgLON5, an IgLON5-fragment or a variant thereof in the subject's
blood and/or b) administering one or more immunosuppressive
pharmaceutical substances.
[0003] Sleep disorders are among the most common complaints seen by
general practitioners. It is estimated that, at any one time,
approximately 30% of the general public are affected by a
significant sleep problem. Persistent sleep loss or poor quality
sleep affects emotional state and behaviour, cognitive function and
performance at school or work, family cohesion and general quality
of life, mental health and also physical wellbeing.
[0004] Sleep disorders are associated with a wide range of
disorders, for example disabilities, psychiatric, neurological,
endocrinological or other medical disorders. Therefore, patients
who have one or more of such symptoms and seek medical attention
are at risk of being misdiagnosed. This is all the more true if the
sleeping disorder is likely to be linked to a neurodegenerative
disease such as Alzheimer's disease or Parkinson's disease, since
clinicians still have to rely, particularly at the early stages of
the diseases, on examining the patient's cognitive abilities, which
are hard to assess in an objective and conclusive manner. Final
confirmation of the diagnosis is possible only through autopsy
after death.
[0005] Whilst reliable biomarkers that may be used for diagnosis at
an early stage or to corroborate differential diagnoses are
available for other types of disorders such as autoimmune metabolic
disorders, in particular autoimmunity in diabetes mellitus, they
remain to be established as a widely accepted tool for the
diagnosis of neurodegenerative diseases or related disorders.
[0006] The importance of an early diagnosis cannot be
overemphasized. Many neurodegenerative disorders, most prominently
Alzheimer's and Parkinson's diseases, cannot be cured, but drugs
are available that may be used to slow down their progression. The
earlier the diagnosis, the better the chances to exploit available
drugs to the full benefit of the patient.
[0007] Notwithstanding the need for a diagnosis at an early stage
of the disease, it is important to bear in mind that the result
needs to be reliable which is not always the case. For example, a
patient was diagnosed as having Alzheimers' disease in a case
reported in the literature, although he actually suffered from the
autoimmune disorder referred to as hashimoto encephalitis. Such
patients may be denied adequate treatment and they continue to
suffer, although administration of commercially available drugs,
such as cortisone in the case of hashimoto encephalitis, would be
likely to ameliorate their symptoms.
[0008] Therefore, a problem underlying the present invention is to
provide an agent and a method for the diagnosis of a disorder
associated with one or more symptoms from the group comprising
parasomnia, stridor, sleep apnea, gait instability, dysarthria,
dysphagia, limb ataxia, vocal cord paralysis, choreic movements in
the limbs and face, memory and attention deficits, apathy,
depressed mood, akathisia and urinary disturbances.
[0009] Another problem underlying the present invention is to
provide a method for diagnosing a neurodegenerative disease
associated with one or more of these symptoms, wherein the method
does not require the clinician in charge to rely on the examination
of the patient's cognitive abilities, preferably a method that
involves a serological assay.
[0010] Another problem underlying the present invention is to
provide a method for diagnosing a novel tauopathy identified by the
inventors that remains to be designated and is associated with one
or more symptoms from the group comprising associated with one or
more symptoms selected from the group comprising parasomnia,
stridor, sleep apnea, gait instability, dysarthria, dysphagia, limb
ataxia, vocal cord paralysis, choreic movements in the limbs and
face, memory and attention deficits, apathy, depressed mood,
akathisia and urinary disturbances.
[0011] Another problem underlying the present invention is to
provide an autoantibody that, when found in a liquid sample taken
from a patient, indicates that said patient is suffering from a
neurodegenerative disease, preferably a tauopathy, more preferably
the novel tauopathy identified by the inventors.
[0012] The problem underlying the present invention is solved by
the subject-matter of the attached independent and dependent
claims.
[0013] In a first aspect the problem underlying the present
invention is solved by 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.
[0014] In a second aspect the problem underlying the present
invention is solved by 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.
[0015] In a third aspect the problem underlying the present
invention is solved by an autoantibody binding to IgLON5, an
IgLON5-fragment or a variant thereof, preferably for use in the
diagnosis of a disease.
[0016] In a fourth aspect the problem underlying the present
invention is solved by a pharmaceutical composition comprising a
polypeptide comprising one or more sequences of IgLON5, an
IgLON5-fragment or a variant thereof.
[0017] In a fifth aspect the problem underlying the present
invention is solved by an in vitro method for diagnosing a disease,
comprising the steps a1) contacting a liquid sample comprising an
antibody from a subject with a polypeptide comprising one or more
sequences of IgLON5, an IgLON5-fragment or a variant thereof, or
a2) contacting a tissue sample comprising IgLON5, an
IgLON5-fragment or a variant thereof from a subject with an
antibody binding to IgLON5, the IgLON5-fragment or the variant
thereof, preferably the autoantibody according the third aspect,
and, following steps a1) or a2), b) detecting formation of a
complex comprising the antibody and IgLON5, the IgLON5-fragment or
the variant thereof, and/or in addition to steps a1)/a2) and b), c)
examining the Human Leukocyte Antigen (HLA) type of the
subject.
[0018] In a first embodiment of the fifth aspect, the complex is
detected using immunofluorescence microscopy or spectroscopy, NMR
spectroscopy, immunodiffusion, mass spectrometry, radioactivity,
chemical crosslinking, surface plasmon resonance, native gel
electrophoresis, chromatography and/or enzymatic activity.
[0019] In a sixth aspect, the problem underlying the present
invention is solved by an in vitro method for diagnosing a disease,
preferably an autoimmune disease, comprising the step of detecting
in a liquid sample comprising antibodies an autoantibody binding to
IgLON5.
[0020] In a seventh aspect the problem underlying the present
invention is solved by a medical or diagnostic device comprising an
autoantibody according to the third aspect of the invention or a
polypeptide comprising one or more sequence of IgLON5, an
IgLON5-fragment or a variant thereof.
[0021] In a eighth aspect the problem underlying the present
invention is solved by a test kit for the diagnosis of a disease,
which test kit comprises the autoantibody according to the third
aspect of the present invention and/or a polypeptide comprising one
or more sequences of IgLON5, an IgLON5-fragment or a variant
thereof.
[0022] In a first embodiment of the both the seventh and the eighth
aspect, the problem is solved by providing the polypeptide
comprising one or more sequences of IgLON5, an IgLON5-fragment or a
variant thereof in an immobilized form.
[0023] In a second embodiment of the eighth aspect, which is also
an embodiment of the first embodiment of the eighth aspect, the
test kit comprises a means for detecting a complex comprising an
antibody and the polypeptide comprising one or more sequences of
IgLON5, an IgLON5-fragment or a variant thereof, preferably,
wherein the antibody is the autoantibody according to the third
aspect of the present invention.
[0024] In a preferred embodiment of any aspect of the present
invention, the disease is selected from the group comprising sleep
disorders, neurodegenerative diseases and hypoventilation,
preferably a neurodegenerative disease, more preferably a
tauopathy.
[0025] In a preferred embodiment of any aspect of the present
invention, the disease is associated with one or more symptoms,
preferably two or more symptoms, more preferably three or more
symptoms, still more preferably four or more symptoms selected from
the group comprising parasomnia, stridor, sleep apnea, gait
instability, dysarthria, dysphagia, limb ataxia, vocal cord
paralysis, choreic movements in the limbs and face, memory and
attention deficits, apathy, depressed mood, akathisia and urinary
disturbances, which symptoms, most preferably, comprise one or more
sleep problems, dysphagia and at least one of memory problems and
depressed mood.
[0026] In a ninth aspect the problem underlying the present
invention is solved by a method for isolating an autoantibody
binding to IgLON5, an IgLON5-fragment or a variant thereof,
comprising the steps a) contacting a sample comprising the
autoantibody with a polypeptide comprising one or more sequences of
IgLON5, the IgLON5-fragment or the variant thereof, which
polypeptide is preferably immobilized, such that a complex is
formed, b) isolating the complex formed in step a), c) dissociating
the complex isolated in step b), and d) separating the autoantibody
from the polypeptide comprising one or more sequences of IgLON5,
the IgLON5-fragment or the variant thereof.
[0027] In a tenth aspect the problem underlying the present
invention is solved by a method for treating a neurological disease
associated with one or more symptoms selected from the group
comprising parasomnia, stridor, sleep apnea, gait instability,
dysarthria, dysphagia, limb ataxia, vocal cord paralysis, choreic
movements in the limbs and face, memory and attention deficits,
apathy, depressed mood, akathisia and urinary disturbancesin a
subject, comprising the steps a) reducing the concentration of
autoantibodies binding to IgLON5, an IgLON5-fragment or a variant
thereof in the subject's blood and/or b) administering one or more
immunosuppressive pharmaceutical substances, preferably selected
from the group comprising rituximab, prednisone,
methylprednisolone, cyclophosphamide, mycophenolatemofetil,
intravenous immunoglobulin, tacrolimus, cyclosporine, methotrexate,
azathioprine and/or the pharmaceutical composition according to the
fourth aspect of the present invention.
[0028] In a preferred embodiment of any aspect of the present
invention, the sequence of IgLON5 is the sequence according to SEQ
ID NO 1 and the sequence of the IgLON5-fragment is a sequence
selected from the group comprising 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 and SEQ ID NO 10 and, preferably, the polypeptide comprises all
of the sequences SEQ ID NO 1 to SEQ ID NO 10.
[0029] In another preferred embodiment of any aspect of the present
invention, the polypeptide comprising one or more sequences of
IgLON5, an IgLON5-fragment or a variant thereof is provided in the
form of a cell comprising a nucleic acid encoding said polypeptide
or in the form of a tissue comprising said polypeptide or variant
thereof.
[0030] The present invention is based on the inventors' surprising
finding that neurodegenerative diseases, in particularly
tauopathies exist that may be associated with the emergence of
autoantibodies binding to IgLON5, an IgLON5-fragment or a variant
thereof.
[0031] Furthermore, the present inventors have surprisingly found
that a new type of tauopathy, exists that is associated with one or
more of symptoms from the group comprising parasomnia, stridor,
sleep apnea, gait instability, dysarthria, dysphagia, limb ataxia,
vocal cord paralysis, choreic movements in the limbs and face,
memory and attention deficits, apathy, depressed mood, akathisia
and urinary disturbances. The inventors have surprisingly found
that autoantibodies binding to IgLON5, an IgLON5-fragment or a
variant thereof may be used to diagnose said disorder.
[0032] Furthermore, the present inventors have surprisingly found
that autoantibodies binding to IgLON5, an IgLON5-fragment or a
variant thereof may be used for the differential diagnosis of
disorders characterised by one or more of symptoms from the group
comprising parasomnia, stridor, sleep apnea, gait instability,
dysarthria, dysphagia, limb ataxia, vocal cord paralysis, choreic
movements in the limbs and face, memory and attention deficits,
apathy, depressed mood, akathisia and urinary disturbances, in
particular neurodegenerative diseases, preferably for
distinguishing the novel tauopathy identified by the inventors from
other types of neurodegenerative diseases, more preferably
tauopathies associated with similar symptoms.
[0033] The present invention centers around a polypeptide
comprising one or more sequences of IgLON5, an IgLON5-fragment or a
variant thereof for use in diagnosis and treatment and antibodies,
in particular autoantibodies binding to IgLON5, an IgLON5-fragment
or a variant thereof. IgLON5 is a member of a family of cell
adhesion molecules involved in the control of neuronal pathfinding
and synaptogenesis (Hashimoto, T., Yamada, M., Maekawa, S.,
Nakashima, T., Miyata, S. (2008) IgLON cell adhesion molecule Kilon
is a crucial modulator for synapse number in hippocampal neurons,
Brain Res 2008; 1224: 1-11). It has been shown that the expression
of IgLON family members affects the proliferation and cell size of
type-1 astrocytes (Sugimoto, C., Morita, S., Miyata, S. (2012)
Overexpression of IgLON cell adhesion molecules changes
proliferation and cell size of cortical astrocytes, Cell Biochem
Funct., 2012 July; 30(5):400-5), but their precise roles and the
mechanisms underlying their actions remain to be elucidated.
[0034] Neither IgLON5 nor autoantibodies binding to IgLON 5 have
been described in the prior art as linked, let alone causatively
linked, to neurological diseases such as tauopathies, let alone
useful for diagnosing them.
[0035] In a preferred embodiment, the polypeptide referred to as
"IgLON5" has the sequence represented by NP_001094842.1, which, as
all sequence accession numbers referred to throughout this
application, refers to the sequence deposited in the NCBI data
base, more specifically the version online on Dec. 4, 2013.
However, the teachings of the present invention may not only be
carried out using polypeptides, in particular a polypeptide
comprising the full-length sequence of IgLON5, having the exact
amino acid sequences referred to in this application explicitly,
for example by name, sequence or accession number, or implicitly,
but also using fragments or variants of such polypeptides.
[0036] The term "fragment", with regard to IgLON5, refers to a less
than full length sequence of said protein, encompassing e.g. an
amino acid sequence which is truncated at one or both termini by
one or more amino acids. Alternatively or in addition, such peptide
sequence may comprise internal deletions of one or more amino
acids. Thereby the residual length of the fragment equals or
exceeds the length of one or more continuous or conformational
epitopes, e.g. 6, 7, 8, 9, 10, 11, 12, 20, 25, 30, 40, 50, 60, 70,
80, 90, 100 or more amino acids.
[0037] The term "variant" of IgLON5 or a fragment thereof relates
to a polypeptide comprising amino acid sequences that are at least
70, 75, 80, 85, 90, 92, 94, 95, 96, 97, 98 or 99% identical to the
reference amino acid, i.e. IgLON5 or a fragment thereof, wherein,
more preferably, amino acids other than those essential for the
biological activity, for example the ability of an antigen to bind
specifically to an antibody, preferably autoantibody, more
preferably an autoantibody binding to IgLON5, an IgLON5-fragment or
a variant thereof from a patient suffering from the novel tauopathy
identified by the inventors, or the fold or structure of the
polypeptide are deleted or substituted, wherein one or more such
essential amino acids may optionally be replaced in a conservative
manner or additional amino acids may be inserted such that the
biological activity of the polypeptide is preserved. The state of
the art comprises various methods that may be used to align two
given nucleic acid or amino acid sequences and to calculate the
degree of identity, see for example Arthur Lesk (2008),
Introduction to bioinformatics, Oxford University Press, 2008, 3rd
edition. In a preferred embodiment, the ClustalW software (Larkin,
M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P.
A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez,
R., Thompson, J. D., Gibson, T. J., Higgins, D. G. (2007). Clustal
W and Clustal X version 2.0. Bioinformatics, 23, 2947-2948) is
used.
[0038] Such variants of IgLON5 and IgLON5-fragments may be prepared
e.g. by introducing deletions, insertions or substitutions in
nucleic acid sequences encoding them, or by chemical synthesis or
modification. Moreover, variants of IgLON5 and IgLON5-fragments may
also be generated by fusion with other known polypeptides or
variants thereof and encompass active portions or domains,
preferably having a sequence identity of at least 70, 75, 80, 85,
90, 92, 94, 95, 96, 97, 98 or 99% when aligned with the active
portion of the reference sequence, wherein the term "active
portion", as used herein, refers to an amino acid sequence, which
is less than the full length amino acid sequence or, in the case of
a nucleic acid sequence, codes for less than the full length amino
acid sequence, respectively, but retains at least some of the
biological activity. For example, an active portion of a protease
is capable of hydrolysing peptide bonds in polypeptides, and an
immunogenic polypeptide retains the ability to bind to an antibody
or autoantibody and, preferably, when administered to mammals,
causes an immune response to occur, more specifically the
production of autoantibodies.
[0039] The term "variant" of a nucleic acid comprises nucleic acids
the complementary strand of which hybridises, preferably under
stringent conditions, to the reference or wild type nucleic acid.
Stringency of hybridisation reactions is readily determinable by
one of ordinary skilled in the art, and in generally is an
empirical calculation dependent on probe length, washing
temperature and salt concentration. In general longer probes
require higher temperatures for proper annealing, while shorter
probes less so. Hybridisation generally depends on the ability of
denatured DNA to reanneal to complementary strands present in an
environment below their melting temperature. The higher the degree
of desired homology between the probe and hybridisable sequence,
the higher the relative temperature which may be used. As a result
it follows that higher relative temperatures would tend to make the
reaction conditions more stringent, while lower temperature less
so. For additional details and explanation of stringency of
hybridisation reactions, see Ausubel, F. M. (1995), Current
Protocols in Molecular Biology. John Wiley & Sons, Inc.
Moreover, the person skilled take in the art may follow the
instructions given in the manual Boehringer Mannheim GmbH (1993)
The DIG System Users Guide for Filter Hybridization, Boehringer
Mannheim GmbH, Mannheim, Germany and in Liebl, W., Ehrmann, M.,
Ludwig, W., and Schleifer, K. H. (1991) International Journal of
Systematic Bacteriology 41: 255-260 on how to identify DNA
sequences by means of hybridisation. In a preferred embodiment,
stringent conditions are applied for any hybridisation, i.e.
hybridisation occurs only if the probe is 70% or more identical to
the target sequence. Probes having a lower degree of identity with
respect to the target sequence may hybridise, but such hybrids are
unstable and will be removed in a washing step under stringent
conditions, for example lowering the concentration of salt to
2.times.SSC or, optionally and subsequently, to 0.5.times.SSC,
while the temperature is, in order of increasing preference,
approximately 50.degree. C.-68.degree. C., approximately 52.degree.
C.-68.degree. C., approximately 54.degree. C.-68.degree. C.,
approximately 56.degree. C.-68.degree. C., approximately 58.degree.
C.-68.degree. C., approximately 60.degree. C.-68.degree. C.,
approximately 62.degree. C.-68.degree. C., approximately 64.degree.
C.-68.degree. C., approximately 66.degree. C.-68.degree. C. In a
particularly preferred embodiment, the temperature is approximately
64.degree. C.-68.degree. C. or approximately 66.degree.
C.-68.degree. C. It is possible to adjust the concentration of salt
to 0.2.times.SSC or even 0.1.times.SSC. Nucleic acid sequences
having a degree of identity with respect to the reference or wild
type sequence of at least 70, 80, 90, 91, 92, 93, 94, 95, 96, 97,
98, 99% may be isolated. In a preferred embodiment, the term
variant of a nucleic acid sequence, as used herein, refers to any
nucleic acid sequence that encodes the same amino acid sequence and
variants thereof as the reference nucleic acid sequence, in line
with the degeneracy of the genetic code. A nucleic acid encoding
IgLON5 has been deposited in the NCBI databases under accession
number NM_001101372.
[0040] The polypeptide comprising one or more sequences of IgLON5,
an IgLON5-fragment or a variant thereof, when used to carry out the
teachings of the present invention, may be provided in any form and
at any degree of purification, from tissues or cells comprising
said polypeptide in an endogenous form, more preferably cells
overexpressing the polypeptide, crude or enriched lysates of such
cells, to purified and/or isolated polypeptide which is essentially
pure. In a preferred embodiment, the polypeptide is a native
polypeptide, wherein the term "native", as used herein, the term
"native polypeptide", as used herein, refers to a folded
polypeptide, more preferably to a folded polypeptide purified from
tissues or cells, more preferably from mammalian cells or tissues,
optionally from non-recombinant tissues or cells. In another
preferred embodiment, the polypeptide is a recombinant protein,
wherein the term "recombinant", as used herein, refers to a
polypeptide produced using genetic engineering approaches at any
stage of the production process, for example by fusing a nucleic
acid encoding the polypeptide to a strong promoter for
overexpression in cells or tissues or by engineering the sequence
of the polypeptide itself. The person skilled in the art is
familiar with methods for engineering nucleic acids and
polypeptides encoded (for example, described in Sambrook, J.,
Fritsch, E. F. and Maniatis, T. (1989), Molecular Cloning, CSH or
in Brown T. A. (1986), Gene Cloning--an introduction, Chapman &
Hall) and for producing and purifying native or recombinant
polypeptides (for example Handbooks "Strategies for Protein
Purification", "Antibody Purification", "Purifying Challenging
Proteins", "Recombinant Protein Purification", "Affinity
Chromatography", "Ion Exchange Chromatography", "Gel Filtration
(Size Exclusion Chromatography)", "Hydrophobic Interaction
Chromatography", "Multimodal Chromatography" (2009/2010), published
by GE Healthcare Life Sciences, and in Burgess, R. R., Deutscher,
M. P. (2009), Guide to Protein Purification). In a preferred
embodiment, a polypeptide is pure if at least 60, 70, 80, 90, 95 or
99 percent of the polypeptide in the respective sample consists of
said polypeptide as judged by SDS polyacrylamide gel
electrophoresis followed by Coomassie blue staining and visual
inspection.
[0041] If the polypeptide comprising one or more sequences of
IgLON5, an IgLON5-fragment or a variant thereof is provided in the
form of tissue, it is preferred that the tissue is from mammalian
brain, for example human rat, primate, donkey, mouse, goat, horse,
sheep or cow brain. If the polypeptide comprising one or more
sequences of IgLON5, an IgLON5-fragment or a variant thereof is
provided in the form of a non-recombinant cell, it is preferred
that the cell is a neuron, preferably a hippocampal neuron or a
cell from the neuropil of a mammal, for example rat, human,
primate, donkey, mouse, goat, horse, sheep or cow. If a cell lysate
is used, it is preferred that the cell lysate comprises the
membranes associated with the surface of the cell. If the
polypeptide comprising one or more sequences of IgLON5, an
IgLON5-fragment or a variant thereof is provided in the form of a
recombinant cell, it is preferred that the recombinant cell is a
eukaryotic cell such as a yeast cell, more preferably a cell from a
multicellular eukaryote such as a plant, mammal, frog or insect,
most preferably from a mammal, for example rat, human, primate,
donkey, mouse, goat, horse, sheep or cow. For example, a HEK293
cell transfected with a nucleic acid functionally encoding the
polypeptide comprising one or more sequences of IgLON5, an
IgLON5-fragment or a variant thereof may be used. The person
skilled in the art is familiar with methods for preparing,
transfecting and culturing such cells, for example those described
in Phelan, M. C. (2001), Basic Techniques in Mammalian Cell Tissue
Culture, John Wiley.
[0042] The polypeptide comprising one or more sequences of IgLON5,
an IgLON5-fragment or a variant thereof used according to the
present invention may be provided in any kind of conformation. For
example, the polypeptide comprising one or more sequences of
IgLON5, an IgLON5-fragment or a variant thereof may be an
essentially unfolded, a partially or a fully folded polypeptide. In
a preferred embodiment, the polypeptide comprising one or more
sequences of IgLON5, an IgLON5-fragment or a variant thereof is
folded in the sense that the IgLON5 epitopes comprised are part of
folded domains and essentially adopt, in a more preferred
embodiment, the fold adopted by the native protein in its natural
environment. The person skilled in the art is familiar with methods
suitable to determine whether or not a polypeptide is folded and if
it is, which structure it has, for example limited proteolysis, NMR
spectroscopy, CD spectroscopy or X-ray crystallography (see for
example Banaszak L. J. (2008), Foundations of Structural Biology,
Academics Press, or Teng Q. (2013), Structural Biology: Practical
Applications, Springer), preferably multidimensional NMR
spectroscopy is used. In a more preferred embodiment, the
polypeptide comprising one or more sequences of IgLON5, an
IgLON5-fragment or a variant thereof is folded such that it adopts
the fold of the native protein in its natural environment and
comprises one or more sequences selected from the group comprising
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 and SEQ ID NO 10 or variants
thereof, preferably all of them. The polypeptide comprising one or
more sequences of IgLON5, an IgLON5-fragment or a variant thereof
may be a fusion protein which comprises amino acid sequences other
than IgLON5, an IgLON5-fragment or a variant thereof, in particular
a C-terminal or N-terminal tag, preferably a C-terminal tag, which
is, in a preferred embodiment, as used herein, an additional
sequence motif or polypeptide having a function that has some
biological or physical function and may, for example, be used to
purify, immobilize, precipitate or identify the polypeptide
comprising one or more sequences of IgLON5, an IgLON5-fragment or a
variant thereof. In a more preferred embodiment, the tag is a
sequence or domain capable of binding specifically to a ligand, for
example a tag selected from the group comprising His tags,
thioredoxin, maltose binding protein, glutathione-S-transferase, a
fluorescence tag, for example from the group comprising green
fluorescent protein (NCBI accession number: AAB51348.1), red
fluorescent protein (AB038175.1), yellow fluorescent protein
(AAA27544.1), orange fluorescent protein (AAP55761.1), blue
fluorescent protein (YP_008577.1) or variants or known functional
fluorescent homologues having other primary sequences thereof and
their variants and/or an epitope tag, for example one selected from
the group comprising myc, flag, HA and V5. In a preferred
embodiment, the polypeptide carries a covalent modification such as
a biotin tag.
[0043] According to the teachings of the present invention, an
antibody, preferably an autoantibody binding to IgLON5, an
IgLON5-fragment or a variant thereof may be used for the diagnosis
of a disease. The person skilled in the art is familiar with
methods for purifying antibodies, for example those described in
Hermanson, G. T., Mallia, A. K., Smith, P. K. (1992), Immobilized
Affinity Ligand Techniques, San Diego: Academic Press. Briefly, an
antigen binding specifically to the antibody of interest, which
antigen is IgLON5, an IgLON5-fragment or a variant thereof, is
immobilized and used to purify, via affinity chromatography, the
antibody of interest from an adequate source. A liquid sample
comprising antibodies from a patient suffering from the novel
tauopathy identified by the inventors may be used as the
source.
[0044] The polypeptide comprising one or more sequences of IgLON5,
an IgLON5-fragment or a variant thereof, when used within the
present invention, may be an immobilized polypeptide. In a
preferred embodiment, the term "immobilized", as used herein,
refers to a molecule bound to a solid carrier insoluble in an
aqueous solution, more preferably via a covalent bond,
electrostatic interactions, encapsulation or entrapment, for
example by denaturing a globular polypeptide in a gel, or via
hydrophobic interactions, most preferably via one or more covalent
bonds. Various suitable carriers, for example paper, metal, silicon
or glass surfaces, microfluidic channels, membranes, beads such as
magnetic beads, column chromatography media, biochips,
polyacrylamide gels and the like have been described in the
literature, for example in Kim, D., Herr, A. E. (2013), Protein
immobilizsation techniques for microfluidic assays,
Biomicrofluidics 7(4), 041501. This way, the immobilized molecule,
together with the insoluble carrier, may be separated from an
aqueous solution in a straightforward manner, for example by
filtration, centrifugation or decanting. An immobilized molecule
may be immobilized in a reversible or irreversible manner. For
example, the immobilization is reversible if the molecule interacts
with the carrier via ionic interactions that can be masked by
addition of a high concentration of salt or if the molecule is
bound via a cleavable covalent bond such as a disulphide bridge
which may be cleaved by addition of thiol-containing reagents. By
contrast, the immobilization is irreversible if the molecule is
tethered to the carrier via a covalent bond that cannot be cleaved
in aqueous solution, for example a bond formed by reaction of an
epoxide group and an amine group as frequently used to couple
lysine side chains to affinity columns. The protein may be
indirectly immobilized, for example by immobilizing an antibody or
other entity having affinity to the molecule, followed by formation
of a complex to the effect that the molecule-antibody complex is
immobilized. Various ways to immobilize molecules are described in
the literature, for example in Kim, D., Herr, A. E. (2013), Protein
immobilizsation techniques for microfluidic assays,
Biomicrofluidics 7(4), 041501. In addition, various reagents and
kits for immobilization reactions are commercially available, for
example from Pierce Biotechnology.
[0045] The polypeptide used to carry out the inventive teachings is
preferably designed such that it is immunogenic, more preferably
such that it binds to autoantibodies binding to IgLON5,
IgLON5-fragments or variants thereof from patients suffering from
the novel tauopathy identified by the inventors. In one embodiment,
such polypeptide comprises a stretch of 6, 7, 8, 9, 10, 11, 12, 20,
25, 30, 40, 50, 60, 70, 80, 90, 100 or more, preferably at least 9
but no more than 16, consecutive amino acids from NP_001094842.1
(IgLON5) or variants thereof. The person skilled in the art is
familiar with guidelines used to design peptides having sufficient
immunogenicity, for example those described in Jackson, D. C.,
Fitzmaurice, C. J., Brown, L. E., Zeng, W. (1999), Preparation and
properties of totally synthetic immunogenes, Vaccine Volume 18,
Issues 3-4, September 1999, Pages 355-361; and Black, M., Trent,
A., Tirrell, M. and Olive, C. (2010), Advances in the design and
delivery of peptide subunit vaccines with a focus on Toll-like
receptor agonists, Expert Rev Vaccines, 2010 February; 9(2):
157-173. Briefly, it is desirable that the peptide meets as many as
possible of the following requirements: (a) it has a high degree of
hydrophilicity, (b) it comprises one or more residues selected from
the group comprising aspartate, proline, tyrosine and
phenylalanine, (c) is has, for higher specificity, no or little
homology with other known peptides or polypeptides, (d) it needs to
be sufficiently soluble and (e) it comprises no glycosylation or
phosphorylation sites unless required for specific reasons.
Alternatively, bioinformatics approaches may be followed, for
example those described by Moreau, V., Fleury, C., Piquer, D.,
Nguyen, C., Novali, N., Villard, S., Laune, D., Granier, C. and
Molina, F. (2008), PEPOP: Computational design of immunogenic
peptides, BMC Bioinformatics 2008, 9:71.
[0046] In a preferred embodiment, the term "diagnosis", as used
herein, refers to any kind of procedure aiming to find out whether
a patient suffers or is likely or more likely than the average
subject to suffer in the future from a disease or disorder in the
past, at the time of the diagnosis or in the future, to find out
how the disease is progressing or is likely to progress in the
future or to evaluate the responsiveness of a patient with regard
to a certain treatment, for example the administration of
immunosuppressive drugs or drugs slowing down the progress of a
neurodegenerative disease. In other words, the term diagnosis
comprises not only diagnosing, but also prognosticating and/or
monitoring the course of a disease or disorder. The person skilled
in the art will appreciate that a clinician does usually not
conclude whether or not the patient suffers or is likely to suffer
from a disease, condition or disorders solely on the basis of a
single diagnostic parameter, but needs to take into account other
aspects, for example the presence of other autoantibodies, markers,
blood parameters, clinical assessment of the patient's symptoms or
the results of medical imaging or other non-invasive methods such
as polysomnography, to arrive at a conclusive diagnosis. See
Baenkler H. W. (2012), General aspects of autoimmune diagnostics,
in Renz, H., Autoimmune diagnostics, 2012, de Gruyter, page 3. The
value of a diagnostic agent or method may also reside the
possibility to rule out one disease, thus allowing for the indirect
diagnosis of another. Therefore, the terms "diagnosis" or does
preferably not imply that the diagnostic methods or agents
according to the present invention will be sufficient to finalize
the diagnosis. In lieu or in addition to the inventive diagnostic
method, one or more of the steps examination by way of
polysomnography or Human Leukocyte Antigen Typing may be performed
to diagnose the disease, preferably the novel tauopathy identified
by the inventors. The person skilled in the art is familiar with
these methods, which are also described in the state of the art,
for example in Kryger, M. H, Roth T, Dement W C (2005), Principles
and Practice of Sleep Medicine (4th ed.). Philadelphia: Elsevier
Saunders, and in Zane, H. D. (2001) Immunology--Theoretical &
Practical Concepts in Laboratory Medicine, W. B. Saunders Company,
in particular in Chapter 14.
[0047] The inventive polypeptide comprising one or more sequences
from IgLON5, an IgLON5-fragment or a variant thereof and/or a
nucleic acid encoding said polypeptide may be used for the
treatment of a disease, for the manufacture of a medicament for
treating a disease and the like. The disease is preferably selected
from the group comprising sleep disorders, neurodegenerative
disease and hypoventilation and is preferably a neurodegenerative
disease, more preferably a tauopathy, most preferably the novel
tauopathy identified by the inventors.
[0048] The polypeptide comprising one or more sequences of IgLON5,
an IgLON5-fragment or a variant thereof may be provided in the form
of a cell comprising and/or expressing a nucleic acid encoding said
polypeptide. If a nucleic acid comprising a sequence that encodes
for the inventive polypeptide or variant thereof is used, such a
nucleic acid may be an unmodified nucleic acid. In a preferred
embodiment, the nucleic acid is a nucleic acid that, as such, does
not occur in nature and comprises, compared to natural nucleic
acid, at least one modification, for example an isotopic content or
chemical modifications, for example a methylation, sequence
modification, label or the like indicative of synthetic origin. In
a preferred embodiment, the nucleic acid is a recombinant nucleic
acid or part or a nucleic acid, and is, in a more preferred
embodiment, part of a vector, in which it may be functionally
linked with a promoter that allows for expression, preferably
overexpression of the nucleic acid. In a preferred embodiment, said
nucleic acid is inside a cell capable of expressing it to the
effect that the inventive polypeptide or a variant thereof is made
and, more preferably, routed to the surface of the cell. Said cell
comprising the nucleic acid encoding the polypeptide comprising one
or more sequences of IgLON5, an IgLON5-fragment or a variant
thereof may be used according to the present invention. The person
skilled in the art is familiar with methods used to synthesize,
modify and amplify such a nucleic acid and to transfect cells using
such a nucleic acid, preferably in a vector that allows for the
transient or permanent maintenance or expression of the nucleic
acid in the cell. The person skilled in the art is also familiar
with suitable vectors, and many of them are commercially available,
for example from Origene. For example, a vector encoding for fusion
constructs with a C-terminal GFP may be used. The cell may be of
eukaryotic or prokaryotic origin and is preferably a mammalian
cell, for example a HEK293 cell. The cell comprising the nucleic
acid encoding for the polypeptide comprising one or more sequences
of IgLON5, an IgLON5-fragment or a variant thereof may be a
recombinant cell or an isolated cell, wherein the term "isolated"
means that the cell is enriched such that, compared to the
environment of the wild type of said cell, fewer cells of other
differentiation or species or in fact no such other cells are
present.
[0049] According to the invention, an antibody, for example an
autoantibody, is provided that is capable of binding specifically
to IgLON5, an IgLON5-fragment or a variant thereof. In a preferred
embodiment, the term "antibody", as used herein, refers to any
immuglobulin-based binding moieties, more preferably one comprising
at least one immunoglobulin heavy chain and one immunoglobulin
light chain, including, but not limited to monoclonal and
polyclonal antibodies as well as variants of an antibody, in
particular fragments, which binding moieties are capable of forming
a complex with an antigen. In a preferred embodiment, the term
"binding specifically", as used herein, means that the binding is
stronger than a binding reaction characterized by a dissociation
constant of 1.times.10.sup.-5 M, more preferably 1.times.10.sup.-7
M, more preferably 1.times.10.sup.-8 M, more preferably
1.times.10.sup.-9 M, more preferably 1.times.10.sup.-10 M, more
preferably 1.times.10.sup.11 M, more preferably 1.times.10.sup.-12
M, as determined by surface plasmon resonance using Biacore
equipment. The antibody may be isolated or in a mixture comprising
further antibodies, polypeptides, metabolites, cells and the like.
In case the antibody is an autoantibody, it may be part of an
autoantibody preparation which is heterogeneous or may be a
homogenous autoantibody, wherein a heterogeneous preparation
comprises a plurality of different autoantibody species as
obtainable by preparation from the sera of human donors, for
example by affinity chromatography using the immobilized antigen to
purify any autoantibody capable of binding to said antigen.
Preferably the antibody is an autoantibody, more preferably an
autoantibody from the IgG class, most preferably from the group of
subclasses comprising IgG1, IgG2 and IgG4, in particular IgG4. The
antibody may be glycosylated or non-glycosylated. The person
skilled in the art is familiar with methods that may be used for
the identification, production and purification of antibodies and
variants thereof, for examples those described in EP 2 423 226 A2
and references therein. The antibody may be used as a diagnostic
agent, by itself, or in combination, for example as a complex, with
the polypeptide comprising IgLON5, an IgLON5-fragment or a variant
thereof.
[0050] The invention provides a pharmaceutical composition
comprising a polypeptide comprising one or more sequences of
IgLON5, an IgLON5-fragment or a variant thereof which is preferably
suitable for administration to a subject, preferably a mammalian
subject, more preferably to a human. Such a pharmaceutical
composition may comprise a pharmaceutically acceptable carrier. The
pharmaceutical composition may, for example, be administered
orally, parenterally, by inhalation spray, topically, by eyedrops,
rectally, nasally, buccally, vaginally or via an implanted
reservoir, wherein the term "parentally", as used herein, comprises
subcutaneous, intracutaneous, intravenous, intramuscular,
intra-articular, intrasynovial, instrasternal, intrathecal,
intralesional and intracranial injection or infusion techniques.
The pharmaceutical composition may be provided in suitable dosage
forms, for example capsules, tablets and aqueous suspensions and
solutions, preferably in sterile form. It may be used in a method
of treatment of a disease, which method comprises administering an
effective amount of the polypeptide comprising IgLON5, an
IgLON5-fragment or a variant thereof to a subject.
[0051] The present invention relates to a complex comprising an
antibody binding to IgLON5, an IgLON5-fragment or a variant
thereof. Such a complex may be used or detected as part of a method
for diagnosing a disease. A liquid sample comprising antibodies
from a subject may be used to practice the method. Such a liquid
sample may be any bodily fluid comprising a representative set of
antibodies from the subject, preferably a sample comprising
antibodies of the IgG immunglobuline class from the subject, more
preferably from the group comprising IgG1, IgG2 and IgG4
subclasses, most preferably from the IgG4 subclass. For example, a
sample may be cerebrospinal fluid (CSF), blood or blood serum,
lymph, insterstitial fluid and is preferably serum or CSF, more
preferably serum.
[0052] In addition, the inventive method may comprise the step
examining the Human Leukocyte Antigen (HLA) type of the subject,
preferably by subjecting a blood sample taken from the patient to
HLA typing. Detection of any allele from the group comprising
HLA-DQB1*0501, HLA-DRB1*1001 and HLA-B27, preferably the
combination of HLA-DQB1*0501 and HLA-DRB1*1001, indicates an
increased risk that the patient suffers or will suffer from a
disease, more preferably a neurological disease, more preferably a
tauopathy, most preferably from the novel tauopathy identified by
the inventors.
[0053] The step contacting a liquid sample comprising antibodies
with a polypeptide comprising one or more sequences of IgLON5, an
IgLON5-fragment or a variant thereof may be carried out by
incubating an immobilized form of said polypeptide in the presence
of the sample comprising antibodies under conditions that are
compatible with the formation of the complex comprising said
polypeptide and an antibody, preferably an autoantibody, binding to
IgLON5, an IgLON5-fragment or a variant thereof. Optionally, the
liquid sample, then depleted of antibodies binding to IgLON5, an
IgLON5-fragment or a variant thereof, may subsequently be removed
to facilitate detection of the complex. Optionally, one or more
washing steps may be contemplated.
[0054] Alternatively, a sample comprising tissue comprising IgLON5,
an IgLON5-fragment or a variant thereof rather than a liquid sample
may be used. The tissue sample is preferably of neuronal origin and
may be a sample of brain tissue, preferably taken from a brain
section selected from the group comprising hypothalamus,
prehypothalamic region and tegmentum of brainstem, more preferably
cerebellum, for example rat cerebellum. Such a sample, which may be
in the form of a tissue section fixed on a carrier, for example a
glass slide for microscopic analysis, may then be contacted with
the inventive antibody, preferably autoantibody, binding to IgLON5,
the IgLON5-fragment or the variant thereof. The antibody is
preferably labeled to allow for distinction from endogenous
antibodies binding to IgLON5, the IgLON5-fragment or the variant
thereof, so that newly formed complexes may be detected and,
optionally, quantified. If the amount of complexes formed is lower
than the amount found in a sample taken from a healthy subject, the
subject from whom the sample examined has been taken is likely to
suffer from a disease.
[0055] Any data demonstrating the presence or absence of the
complex comprising the antibody and IgLON5, the IgLON5-fragment or
the variant thereof may be correlated with reference data. For
example, detection of said complex indicates that the patient who
provided the sample analysed suffered, suffers or is likely to
suffer in the future from the novel tauopathy identified by the
inventors. If a patient has been previously diagnosed, the amount
of complex detected in both runs may be correlated to find out
about the progression of the disease and/or the success of a
treatment. For example, if the amount of complex is found to
increase, it may be concluded that the diseases is progressing
and/or that any treatment attempted is unsuccessful.
[0056] The complex comprising the antibody and IgLON5, the
IgLON5-fragment or the variant thereof may be detected using a
variety of methods known to the person skilled in the art, for
example immunofluorescence microscopy or spectroscopy,
luminescence, NMR spectroscopy, immunodiffusion, radioactivity,
chemical crosslinking, surface plasmon resonance, native gel
electrophoresis or enzymatic activity. While some of these methods
allow for the direct detection of the complex, it is preferred that
one of the binding partners, preferably the antibody or, more
preferably, a second antibody binding to the antibody, to IgLON5,
the IgLON5-fragment or the variant thereof or to the polypeptide
comprising one or more sequences of IgLON5, an IgLON5-fragment or a
variant thereof, is labeled such that the complex may be detected
specifically owing to intrinsic properties of the label, for
example fluorescence, radioactivity, enzymatic activity, visibility
in NMR or MRI spectra or the like. In a preferred embodiment the
diagnosis or prognosis is carried out using a method selected from
the group comprising western blot, dot blot, protein microarray,
ELISA, line blot radioimmune assay and indirect immunofluorescence
microscopy. Alternatively, more than one of these methods may be
used in a complementary manner for more reliable results.
[0057] In a preferred embodiment, the prognosis, diagnosis, methods
or test kit according to the present invention relates to a
diagnostic method selected from the group comprising
immunodiffusion techniques, immunoelectrophoretic techniques, light
scattering immunoassays, light scattering immunoassays,
agglutination techniques, labeled immunoassays such as those from
the group comprising radiolabeled immunoassay, enzyme immunoassays,
chemiluminscence immunoassays, and immunofluorescence techniques.
The person skilled in the art is familiar with these methods, which
are also described in the state of the art, for example in Zane, H.
D. (2001), Immunology--Theoretical & Practical Concepts in
Laboratory Medicine, W. B. Saunders Company, in particular in
Chapter 14.
[0058] In a preferred embodiment, a line blot is used to carry out
the diagnostic method according to the invention. The person
skilled in the art is familiar with the experimental setup, which
is described in the state of the art (Raoult, D., and Dasch, G. A.
(1989), The line blot: an immunoassay for monoclonal and other
antibodies. Its application to the serotyping of gram-negative
bacteria. J. Immunol. Methods, 125 (1-2), 57-65; WO2013041540).
Briefly, the one or more antigen of interest, in the case of the
present invention the polypeptide comprising one or more sequences
of IgLON5, an IgLON5-fragment or a variant thereof, may be attached
to a carrier, for example nitrocellulose membrane, often in
combination with additional antigens and controls. The
nitrocellulose carrier is subsequently exposed to a sample
comprising antibodies such as diluted serum. If the sample
comprises an antibody binding to the antigen, a complex is formed
which may be detected, preferably by incubation with a secondary
antibody binding to the constant region of the first antibody,
which secondary antibody comprises a detectable label, for example
a radioactive isotope, a fluorescent dye or, in a preferred
embodiment, an active enzyme fused or linked to the secondary
antibody, such as alkaline phosphatase, which may be readily
assayed using chromogenic substrates followed by simple visual
examination. Suitable reagents, devices and software packages are
commercially available, for example from EUROIMMUN, Lubeck,
Germany.
[0059] In another preferred embodiment, the prognosis, diagnosis,
methods or test kit in line with the inventive teachings
contemplate the use of indirect immunofluorescence. The person
skilled in the art is familiar with such techniques and the
preparation of suitable samples, which are described in the state
of the art (U.S. Pat. No. 4,647,543, Voigt, J., Krause, C.,
Rohwader, E, Saschenbrecker, S., Hahn, M., Danckwardt, M., Feirer,
C., Ens, K, Fechner, K, Barth, E, Martinetz, T., and Stocker, W.
(2012), Automated Indirect Immunofluorescence Evaluation of
Antinuclear Autoantibodies on HEp-2 Cells," Clinical and
Developmental Immunology, vol. 2012, doi:10.1155/2012/65105;
Bonilla, E., Francis, L., Allam, F., et al., "Immunofluorescence
microscopy is superior to fluorescent beads for detection of
antinuclear antibody reactivity in systemic lupus erythematosus
patients," Clinical Immunology, vol. 124, no. 1, pp. 18-21, 2007).
Briefly, a carrier, such as a cover glass for use in microscopy, is
coated with cells or tissue sections comprising the antigen, in the
case of the present invention the polypeptide comprising one or
more sequences of IgLON5, an IgLON5-fragment or a variant thereof.
The carrier comprising the antigen is exposed to a patient sample
comprising antibodies such as diluted serum. If the sample
comprises an antibody binding to the antigen, the resulting complex
may be detected, preferably by incubation with a secondary antibody
comprising a fluorescent dye such as fluorescein, followed by
visual examination using fluorescence microscopy. Suitable
reagents, devices and software packages are commercially available,
for example from EUROIMMUN, Lubeck, Germany.
[0060] Within the scope of the present invention, a medical or
diagnostic device comprising an autoantibody or a polypeptide
comprising one or more sequence of IgLON5, an IgLON5-fragment or a
variant thereof according to the present invention is provided.
Preferably such a medical or diagnostic device comprises the
polypeptide comprising one or more sequences of IgLON5, an
IgLON5-fragment or a variant thereof in a form that allows
contacting it with an aqueous solution, more preferably the liquid
human sample, in a straightforward manner. In particular, the
polypeptide comprising one or more sequences of IgLON5, an
IgLON5-fragment or a variant thereof may be immobilized on the
surface of a carrier, which carrier comprises, but is not limited
to glass plates or slides, biochips, microtiter plates, beads, for
example magnetic beads, chromatography columns, membranes or the
like. Exemplary medical devices include line blots, microtiter
plates and biochips. In addition to the inventive polypeptide
comprising one or more sequences of IgLON5, an IgLON5-fragment or a
variant thereof, the medical or diagnostic device may comprise
additional polypeptides, for example positive or negative controls
or known other antigens binding to autoantibodies of diagnostic
value, particularly those related other diseases associated with
one or more symptoms selected from the group comprising parasomnia,
stridor, sleep apnea, gait instability, dysarthria, dysphagia, limb
ataxia, vocal cord paralysis, choreic movements in the limbs and
face, memory and attention deficits, apathy, depressed mood,
akathisia and urinary disturbances, which diseases are preferably
selected from the group comprising sleep disorders,
neurodegenerative disease and hypoventilation.
[0061] The inventive teachings provide a kit for diagnosing a
disease associated with one or more symptoms selected from the
group comprising stridor, sleep apnea, gait instability,
dysarthria, dysphagia, limb ataxia, vocal cord paralysis, choreic
movements in the limbs and face, memory and attention deficits,
apathy, depressed mood, akathisia and urinary disturbances.
Preferably the disease is selected from the group comprising sleep
disorders, neurodegenerative diseases and hypoventilation. Such a
kit may comprise instructions detailing how to use the kit and a
means for contacting the polypeptide comprising one or more
sequences of IgLON5, an IgLON5-fragment or a variant thereof with a
bodily fluid sample from a subject, preferably a human subject, for
example a line blot, wherein the polypeptide comprising one or more
sequences of IgLON5, an IgLON5-fragment or a variant thereof is
immobilized on the line blot. Furthermore, the kit may comprise a
positive control, for example a batch of autoantibody or
recombinant antibody known to bind to IgLON5, an IgLON5-fragment or
a variant thereof and a negative control, for example a protein
having no detectable affinity to IgLON5, an IgLON5-fragment or a
variant thereof such as bovine serum albumin. Finally, such a kit
may comprise a standard solution for preparing a calibration
curve.
[0062] In a preferred embodiment the kit comprises a means for
detecting a complex comprising the polypeptide comprising one or
more sequences of IgLON5, an IgLON5-fragment or a variant thereof
and an antibody binding to IgLON5, an IgLON5-fragment or a variant
thereof. Such means is preferably an agent that binds to said
complex and modifies the complex or carries a label such that makes
the complex detectable. For example, said means may be a labeled
secondary antibody binding to said polypeptide, at a binding site
other than the binding site recognized by the primary antibody or
to a constant region of the primary antibody. Alternatively, said
means may be a crosslinking reagent chemically linking the antibody
and the polypeptide comprising one or more sequences of IgLON5, an
IgLON5-fragment or a variant thereof, so the complex may be
identified on account of its increased molecular weight, for
example by gel electrophoresis or size-exclusion chromatography
[0063] The present invention provides a method for isolating an
antibody, preferably an autoantibody, binding to a polypeptide
comprising one or more sequences of IgLON5, an IgLON5-fragment or a
variant thereof, comprising the steps a) contacting a sample
comprising the antibody with a polypeptide comprising one or more
sequences of IgLON5, an IgLON5-fragment or a variant thereof such
that a complex is formed, b) isolating the complex formed in step
a), c) dissociating the complex isolated in step b), and d)
separating the antibody from the polypeptide comprising one or more
sequences of IgLON5, an IgLON5-fragment or a variant thereof. A
sample from a patient suffering from the novel tauopathy identified
by the inventors may be used as the source of antibody.
Alternatively the antibody may be a recombinant antibody. It is
preferred that the polypeptide is immobilized, for example on the
matrix of a column suitable for affinity chromatography or on a
magnetic bead, since it is straightforward to separate the complex
comprising the polypeptide and the antibody in step b) if such is
the case. Subsequently, the antibody may be separated from the
immobilized antigen in step c), for example by eluting the antibody
by addition of an excess of non-immobilized antigen or by adding an
agent interfering with intramolecular interactions, for example
guanidinium chloride or sodium chloride at a high concentration,
the latter if that electrostatic interactions are essential to
maintain the complex. The person skilled in the art is familiar
with methods to carry out each of these steps. Suitable methods are
described in the state of the art, for example in the Handbooks
"Affinity chromatography", "Strategies for Protein Purification"
and "Antibody Purification" (2009/2010), published by GE Healthcare
Life Sciences.
[0064] The inventions provides a method for treating the novel
tauopathy identified by the inventors in a subject, comprising the
steps a) reducing the concentration of autoantibodies binding to
IgLON5, an IgLON5-fragment or a variant thereof in the subject's
blood and/or b) administering one or more immunosuppressive
pharmaceutical substances, preferably selected from the group
comprising rituximab, prednisone, methylprednisolone,
cyclophosphamide, mycophenolatemofetil, intravenous immunoglobulin,
tacrolimus, cyclosporine, methotrexate, azathioprine and/or the
pharmaceutical composition according to the present invention,
wherein any other immunosuppressive pharmaceutical substances that
may become available in the future may be used in lieu of or in
addition to the substances specifically mentioned above. The
concentration of autoantibodies binding to IgLON5, an
IgLON5-fragment or a variant thereof may be reduced by obtaining
from the patient blood, bringing it into contact with the
polypeptide comprising one or more sequences of IgLON5, an
IgLON5-fragment or a variant thereof, followed by separating and
readministrating to the patient the blood depleted of
autoantibodies binding to IgLON5, an IgLON5-fragment or a variant
thereof. It is preferred that the polypeptide comprising one or
more sequences of IgLON5, an IgLON5-fragment or a variant thereof
is immobilized and the blood removed from the patient contacted
with said immobilised polypeptide and readministered to the patient
in a continuous manner. Alternatively the polypeptide comprising
one or more sequences of IgLON5, an IgLON5-fragment or a variant
thereof or the pharmaceutical composition according to the present
invention may be administered to the patient to the effect that
autoantibodies circulating in the patients' body form a complex
with the administered complex rather than targeting endogenous
IgLON5, IgLON5-fragments or variants thereof.
[0065] The person skilled in the art will appreciate that any of
the preferred embodiments discussed throughout this application may
be applied to any of the aspects of the inventions.
[0066] The present invention is further illustrated by the
following figures and non-limiting sequences and examples from
which further features, embodiments, aspects and advantages of the
present invention may be taken.
[0067] 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 02.
Warmer colours indicate more dominant frequencies, and cooler
colours indicate frequencies that are not as dominant.
Abbreviations: Rems Rapid eye movements; RBD: REM sleep behavior
disorder; PLM: Periodic limb movements; RPLM: Rapid periodic leg
movements.
[0068] 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.
[0069] 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 .mu.m, C=200 .mu.m, D=50
.mu.m and E=20 .quadrature..mu.m.
[0070] 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 IgG1 (A), IgG2 (B),
IgG3 (C), and IgG4 (D). Robust neuropil immunostaining is only
observed with IgG4. Scale bar=200.quadrature..mu.m.
[0071] 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 (red) that
specifically express IgLON5, as demonstrated by the EGFP
fluorescence (green). Both reactivities are shown merged in C.
Nuclei counterstained with DAPI. Scale bar=20 .mu.m.
[0072] 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).
[0073] 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, IP100367494) (14% of
protein coverage in A and 29% in B).
[0074] 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 .mu.m, C and D=.quadrature.20
.mu.m.
[0075] Throughout this application, a number of sequences are
disclosed which are referred to as:
[0076] SEQ ID NO 1: human IgLON5
[0077] SEQ ID NO 2: rodent/human IgLON5 fragment, see FIG. 7
[0078] SEQ ID NO 3: human IgLON5 fragment
[0079] SEQ ID NO 4: rodent/human IgLON5 fragment, see FIG. 7
[0080] SEQ ID NO 5: rodent/human IgLON5 fragment, see FIG. 7
[0081] SEQ ID NO 6: rodent/human IgLON5 fragment, see FIG. 7
[0082] SEQ ID NO 7: rodent/human IgLON5 fragment, see FIG. 7
[0083] SEQ ID NO 8: rodent/human IgLON5 fragment, see FIG. 7
[0084] SEQ ID NO 9: rodent/human IgLON5 fragment, see FIG. 7
[0085] SEQ ID NO 10: rodent/human IgLON5 fragment, see FIG. 7
[0086] SEQ ID NO 11: rodent IgLON5 fragment, see FIG. 7
[0087] SEQ ID NO 12: artificial IgLON5 construct, see FIG. 7
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EXAMPLES
Patients and Methods
[0133] Inclusion Criteria, Patients, and Controls
[0134] 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 Biomediques August Pi i
Sunyer, Barcelona, Spain.
[0135] Polysomnographic (PSG) Studies
[0136] 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.
[0137] 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 activity--e.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.
[0138] 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., Hogl
B., Trenkwalder C., Bliwise D. (2011), Scoring sleep in
neurological patients: the need for specific considerations, Sleep
2011; 34:1283-4).
[0139] Procedures for Detection of IgLON5 Antibodies and
Characterization of the Antigen
[0140] Female Wistar rats were euthanized and the brain was
removed, sagittally sectioned, immersed in 4% paraformaldehyde at
4.degree. 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 autoantigens--pathogenesis, 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).
[0141] 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.degree. 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.
[0142] 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.degree. 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, Oreg.). 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, Clin Neurophysiol 2002;
113:552-60).
[0143] Immunoprecipitation experiments were done with cultures of
rat hippocampal neurons grown in 100 mm wells, and incubated at
37.degree. 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.degree. C. The
supernatant was retained, incubated with protein NG agarose beads
(20423; Pierce, Rockford, Ill.) overnight at 4.degree. 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.
[0144] 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.degree. 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, Oreg.) (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).
[0145] 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.
[0146] Neuropathological Studies
[0147] 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., Llado A., Clarimon 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., Llado A., Clarimon 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.
[0148] Results
[0149] Clinical Findings
[0150] 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.
[0151] 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).
[0152] Polysomnographic Studies
[0153] 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.
[0154] 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).
[0155] Antibody Characterization
[0156] 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.
[0157] 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.
[0158] Identification of IgLON Family Member 5 as the Targeted
Antigen
[0159] 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.
[0160] 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).
[0161] Neuropathological Examination
[0162] 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.
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
Akathisia. 3 cycles of iv M/59 problems (insidious) behaviors.*
Stridor with paresis and attention Hypersalivation. steroids Ig, Cy
(chronic) OSA. Fragmented sleep. complaints. Episodes of intense No
change Occasional confusional Apathy, low perspiration. awakenings.
mood ** Intermittent intense EDS Patient 2 Sleep 6 years Sleep
movements and Normal Mild dysphagia Mild memory Akathisia 3 cycles
of iv M/53 problems (insidious/ behaviors.* Stridor with and
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
dysphagia Mild memory Akathisia 3 cycles of iv M/52 problems
(insidious/ behaviors.* Stridor with unsteadiness and dysarthria.
and attention Chorea steroids Ig, Cy (subacute) fluctuating) OSA.
Intense episodic infrequent Bilateral VC complaints. No change EDS
falls paresis Apathy, low mood 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 sleep.
central Anxiety andbradycardia. Sudden death Nocturnal confusional
hypoventilation Mild vertical gaze awakenings. palsy Moderate EDS
Patient 5 Gait 6 months Sleep movements and Gait failure Dysphagia.
Depressed Mild oculomotor Iv/oral F/76 instability (rapidly
behaviors* with severe Bilateral VC mood dysfunction with steroids
with falls progressive) Stridor with OSA postural paresis. Central
saccadic intrusions No change (subacute) instability
hypoventilation on pursuit Sudden death while asleep Patient 6 Gait
2 months Frequent sleep Gait failure Dysphagia. Long history of
Bilateral horizontal Iv steroids, Ig, F/65 difficulties (rapidly
movements and with severe Mandibular severe chronic nystagmus. Limb
and rituximab (subacute) progressive) behaviors. Loud postural
spasms. Central depression ataxia. Improved.*** "snoring" with OSA.
instability hypoventilation Sudden death Fragmented sleep during
daytime Patient 7 Gait 12 years Frequent sleep Cerebellar Dysphagia
Mental Chorea Iv/oral F/59 instability (slowly movements and
ataxia. Dysarthria slowness with Bradykinesia steroids; with falls
progressive) behaviors. Loud Severe memory and Limb ataxia
rituximab (chronic) "snoring". Mild EDS postural attention No
change instability difficulties Sudden death Depressed during mood
daytime Patient 8 Gait 5 years Frequent sleep Cerebellar Dysphagia
Mild memory Chorea Iv Ig F/58 instability (slowly movements and
ataxia Dysarthria and attention Vertical/horizontal No change with
falls progressive) behaviors. Central complaints nystagmus with
Dead from (chronic) Stridor with OSA. hypoventilation Depressed
saccadic intrusions central Fragmented sleep. mood Limb dysmetria
hypoventilation 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
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
TABLE-US-00003 TABLE 3 Topographical distribution of neuronal loss
and tau pathology Patient 2 (tablet) Patient 5 (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 + ++ + +++ 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 200x or 400x
magnification.
Sequence CWU 1
1
121336PRThuman 1Met Pro Pro Pro Ala Pro Gly Ala Arg Leu Arg Leu Leu
Ala Ala Ala 1 5 10 15 Ala Leu Ala Gly Leu Ala Val Ile Ser Arg Gly
Leu Leu Ser Gln Ser 20 25 30 Leu Glu Phe Asn Ser Pro Ala Asp Asn
Tyr Thr Val Cys Glu Gly Asp 35 40 45 Asn Ala Thr Leu Ser Cys Phe
Ile Asp Glu His Val Thr Arg Val Ala 50 55 60 Trp Leu Asn Arg Ser
Asn Ile Leu Tyr Ala Gly Asn Asp Arg Trp Thr 65 70 75 80 Ser Asp Pro
Arg Val Arg Leu Leu Ile Asn Thr Pro Glu Glu Phe Ser 85 90 95 Ile
Leu Ile Thr Glu Val Gly Leu Gly Asp Glu Gly Leu Tyr Thr Cys 100 105
110 Ser Phe Gln Thr Arg His Gln Pro Tyr Thr Thr Gln Val Tyr Leu Ile
115 120 125 Val His Val Pro Ala Arg Ile Val Asn Ile Ser Ser Pro Val
Thr Val 130 135 140 Asn Glu Gly Gly Asn Val Asn Leu Leu Cys Leu Ala
Val Gly Arg Pro 145 150 155 160 Glu Pro Thr Val Thr Trp Arg Gln Leu
Arg Asp Gly Phe Thr Ser Glu 165 170 175 Gly Glu Ile Leu Glu Ile Ser
Asp Ile Gln Arg Gly Gln Ala Gly Glu 180 185 190 Tyr Glu Cys Val Thr
His Asn Gly Val Asn Ser Ala Pro Asp Ser Arg 195 200 205 Arg Val Leu
Val Thr Val Asn Tyr Pro Pro Thr Ile Thr Asp Val Thr 210 215 220 Ser
Ala Arg Thr Ala Leu Gly Arg Ala Ala Leu Leu Arg Cys Glu Ala 225 230
235 240 Met Ala Val Pro Pro Ala Asp Phe Gln Trp Tyr Lys Asp Asp Arg
Leu 245 250 255 Leu Ser Ser Gly Thr Ala Glu Gly Leu Lys Val Gln Thr
Glu Arg Thr 260 265 270 Arg Ser Met Leu Leu Phe Ala Asn Val Ser Ala
Arg His Tyr Gly Asn 275 280 285 Tyr Thr Cys Arg Ala Ala Asn Arg Leu
Gly Ala Ser Ser Ala Ser Met 290 295 300 Arg Leu Leu Arg Pro Gly Ser
Leu Glu Asn Ser Ala Pro Arg Pro Pro 305 310 315 320 Gly Leu Leu Ala
Leu Leu Ser Ala Leu Gly Trp Leu Trp Trp Arg Met 325 330 335
222PRTartificialrodent/human IgLON5fragment 2Gly Gln Ala Gly Glu
Tyr Glu Cys Val Thr His Asn Gly Val Asn Ser 1 5 10 15 Ala Pro Asp
Ser Arg Arg 20 311PRTartificialhuman IgLON5 fragment 3Leu Leu Ser
Ser Gly Thr Ala Glu Gly Leu Lys 1 5 10 416PRTartificialrodent/human
IgLON5 fragment 4Ser Asn Ile Leu Tyr Ala Gly Asn Asp Arg Trp Thr
Ser Asp Pro Arg 1 5 10 15 517PRTartificialrodent/human IgLON5
fragment 5Asp Gly Phe Thr Ser Glu Gly Glu Ile Leu Glu Ile Ser Asp
Ile Gln 1 5 10 15 Arg 621PRTartificialrodent/human IgLON5 fragment
6Gly Gln Ala Gly Glu Tyr Glu Cys Val Thr His Asn Gly Val Asn Ser 1
5 10 15 Ala Pro Asp Ser Arg 20 717PRTartificialrodent/human IgLON5
fragment 7His Gln Pro Tyr Thr Thr Gln Val Tyr Leu Ile Val His Val
Pro Ala 1 5 10 15 Arg 811PRTartificialrodent/human IgLON5 fragment
8Ser Met Leu Leu Phe Ala Asn Val Ser Ala Arg 1 5 10
910PRTartificialrodent/human IgLON5 fragment 9Ser Asn Ile Leu Tyr
Ala Gly Asn Asp Arg1 5 10 1018PRTartificialrodent/human IgLON5
fragment 10Val Leu Val Thr Val Asn Tyr Pro Pro Thr Ile Thr Asp Val
Thr Ser 1 5 10 15 Ala Arg 1111PRTartificialrodent IgLON5 fragment
11Leu Leu Ser Ser Gly Ser Ala Glu Gly Leu Lys 1 5 10
12344PRTartificialartificial IgLON5 construct 12Met Asp Ala Tyr Phe
Thr Glu Cys Ile Pro Ser Lys Thr Asn Lys Arg 1 5 10 15 Tyr Phe Tyr
Asn Val Cys Val Thr Ala Leu Ala Gly Leu Ala Val Ile 20 25 30 Ser
Arg Gly Leu Leu Ser Gln Ser Leu Glu Phe Ser Ser Pro Ala Asp 35 40
45 Asn Tyr Thr Val Cys Glu Gly Asp Asn Ala Thr Leu Ser Cys Phe Ile
50 55 60 Asp Glu His Val Thr Arg Val Ala Trp Leu Asn Arg Ser Asn
Ile Leu 65 70 75 80 Tyr Ala Gly Asn Asp Arg Trp Thr Ser Asp Pro Arg
Val Arg Leu Leu 85 90 95 Ile Asn Thr Pro Glu Glu Phe Ser Ile Leu
Ile Thr Gln Val Gly Leu 100 105 110 Gly Asp Glu Gly Leu Tyr Thr Cys
Ser Phe Gln Thr Arg His Gln Pro 115 120 125 Tyr Thr Thr Gln Val Tyr
Leu Ile Val His Val Pro Ala Arg Ile Val 130 135 140 Asn Ile Ser Ser
Pro Val Ala Val Asn Glu Gly Gly Asn Val Asn Leu 145 150 155 160 Leu
Cys Leu Ala Val Gly Arg Pro Glu Pro Thr Val Thr Trp Arg Gln 165 170
175 Leu Arg Asp Gly Phe Thr Ser Glu Gly Glu Ile Leu Glu Ile Ser Asp
180 185 190 Ile Gln Arg Gly Gln Ala Gly Glu Tyr Glu Cys Val Thr His
Asn Gly 195 200 205 Val Asn Ser Ala Pro Asp Ser Arg Arg Val Leu Val
Thr Val Asn Tyr 210 215 220 Pro Pro Thr Ile Thr Asp Val Thr Ser Ala
Arg Thr Ala Leu Gly Arg 225 230 235 240 Ala Ala Leu Leu Arg Cys Glu
Ala Met Ala Val Pro Pro Ala Asp Phe 245 250 255 Gln Trp Tyr Lys Asp
Asp Arg Leu Leu Ser Ser Gly Ser Ala Glu Gly 260 265 270 Leu Lys Val
Gln Thr Glu Arg Thr Arg Ser Met Leu Leu Phe Ala Asn 275 280 285 Val
Ser Ala Arg His Tyr Gly Asn Tyr Thr Cys Arg Ala Ala Asn Arg 290 295
300 Leu Gly Ala Ser Ser Ala Ser Met Arg Leu Leu Arg Pro Gly Ser Leu
305 310 315 320 Glu Asn Ser Ala Pro Arg Pro Pro Gly Pro Leu Thr Leu
Leu Ser Ala 325 330 335 Leu Ser Trp Leu Trp Trp Arg Met 340
* * * * *