Transcript Rush Storz - USD Biology

Effects of H1N1
Influenza on the sleepwake regulatory neurons.
Or: Mr. Bean goes on vacation, and gets narcolepsy, a tale two possible
tragedies.
By. C. Rush Storz
Narcolepsy: a clinical review
• Dr. Guy Leschziner
History
• First described in the
17th century.
• Coined “narcolepsy”
towards the end of the
19th century.
• 1960: Vogel discovered
the association with
sleep-onset rapid eye
movement periods
(SOREMP).
History Cont.
• Identification of
narcolepsy with cataplexy
in dogs in the 1970’s led to
the discovery of mutations
in the hypocretin-2
receptor, and first
implicated the role of
hypocretin (orexin).
Pathophysiology
• Strong genetic component• Familial studies that show risk of
developing in a first degree relative to
be 1-2%.
• Strong human leucocyte antigen
(HLA) associated with HLA
DQB1*0602.
• 98% of patients that have narcolepsy
with cataplexy have this.
Pathophysiology Cont.
• Suggestions of infective triggers:
• Winter infections: high serum titers
of anti-streptolysin O antibodies.
• H1N1 flu and/or vaccine
• Recent attention focused on antiTribbles 2 analogue antibody.
Pathophysiology
cont.
• Peptides hypocretin-1 and
hypocretin-2, only found
in the lateral
hypothalamus.
• Patients with narcolepsy
w/ cataplexy show loss
~70000 hypocretin
producing neurons.
Pathophysiology cont.
• Low & absent levels of hypocrexin1 in CSF are found in patients w/
narcoplexy with cataplexy.
• Not as much in patients with only
narcolepsy.
Pathophysiology Cont.
• Hypocretin-producing neurons
stimulate histaminergic neurons in:
• Tubermamillary nucleus
• Locus coeruleus
• Raphe nuclei
• Basal forebrain
Pathophysiology Cont.
• Hypocretinergic neurons also have
excitatory projections:
• Ventrolateral periaqueductal grey
• Lateral pontine tegmentum
• Loss of hypocretinergic neurons
causes inherent instability area that
maintains wakefulness and
preventing REM sleep.
Clinical Features
• Affects 1 in 3000 people.
• Bimodial distrubution
• Peaks of onset between 15 and 36 years
old.
• Only mandatory feature of narcolepsy
is excessive daytime sleepiness on an
almost daily basis for at least 3 months.
• All graduate, law, and medical students?
Clinical Features Cont.
Narcolepsy with cataplexy
Narcolepsy without cataplexy
• Patient complains of excessive sleepiness.
• Definite history of cataplexy—triggered
• Patient complains of excessive sleepiness.
• Cataplexy is not present, there may be
by emotions.
• Should be confirmed by MSLT; mean
sleep latency on MSLT, with 2 or more
periods of SOREMP’s.
• Not better explained by another sleep
disorder or neurological disorder.
cataplexy-like episodes.
• Must be confirmed by MSLT; mean sleep
latency on MSLT, with 2 or more periods
of SOREMP’s.
• Not better explained by another sleep
disorder or neurological disorder.
Clinical Features Cont.
Other clinical features
• Dreaming in short naps
• Able to fall asleep for seconds or
minutes, and find them to be
refreshing.
• Often wake at night feeling extremely
refreshed and unable to return to
sleep.
• 30-60% of patients have hypnogogic
hallucinations.
Symptomatic Narcolepsy
• May result from other brain
pathology.
• Multiple sclerosis
• Head trauma
• Hypothalamic tumor
• Vascular lesions
• Forms of encephalitis
• Other diseases
Diagnostic Testing
• Mean Sleep Latency Test (MSLT)
• A biological test with high false-
positive and false-negative rate.
• 6% of males and 1.5% of normal
females have an MSLT that tests
positive for narcolepsy.
• Polysomnography that shows REM
sleep arising from wake stage or stage
1 non-REM sleep during the first stage
or REM naps during the daytime.
Diagnostic Testing Cont.
• Low CSF of hypocretin-1 helps
diagnose narcolepsy with cataplexy
but not narcolepsy without
cataplexy.
• DQB1*0602 haplotype is common
in ~95% of patients with cataplexy
but only 40-60% without.
• General population without
narcolepsy: 20-25%
H1N1 Swine flu
• First 6 cases of vaccine induced
narcolepsy tested positive for the
DQB1*0602 haplotype.
• There may also be a link between
H1N1 influenza itself and narcolepsy.
Autoantibody targets in vaccineassociated narcolepsy.
Anna Haggmark-Manberg, Arash Zandian, Bjorn Forsstrom, Mohsen Khademi,
Izaura Lima Bomfim, Cecilia Hellstrom, Lisen Arnheim-Dahlstrom, Tove Hallbook,
Niklas Darin, Ingrid E. Lundberg, Mathias Uhlen, Markku Partinen, Jochen M.
Schwenk, Tomas Olsson, and Peter Nilsson.
Or, Mr. Bean knows that he
is traveling into a H1N1
danger zone and gets his
vaccination before the trip.
Background
• In several Scandanavian countries
incidence of narcolepsy increased after
H1N1 vaccinations in 2009.
• Initial Hypothesis: cross-reactivity
reactions.
• Other studies show evidence of B-cell
autoimmunity recognizing TRIB2.—
passive transfer of IgG to mice.
Other potential targets:
• Alpha-melanocyte-stimulation
hormone;
• Neuropeptide-glutamic acidisoleucine (NEI).
Materials and Methods
• Blood was collected and samples were
prepared as a serum or EDTA plasma.
• Finland: 57 samples were gathered
• 39 narcolepsy
• 19 m, 19 f, 1 missing info
• 20 vaccine-associated
• 16 sporadic
• 18 controls
Materials and Methods Cont.
• Sweden: 176 samples
• 59 narcolepsy
• 29 M; 27 F; 2 missing info.
• 46 displayed vaccine-associated
narcolepsy
Planar antigen microarrays
• Protein Microarrays were developed within the
Human Protein Atlas project.
• 80 AA expressed with a His6- -albumin binding protein
(His6- -ABP) tag.
• Utilized for screening of autoimmunity repertoire in
serum from Finnish narcolepsy patients and controls.
Suspension bead arrays
• This selected and
immobilized on magnetic
beads antigens with higher
reactivity in narcolepsy
samples.
• Also differentiated between
vaccine-associated and
sporadic disease.
• Samples were immobilized
with anti-His6-ABP
antibody
• Antigens 5x reactivity
frequency were selected.
Peptide Arrays
• One sample that was highly reactive
to several antigens was selected.
• Used for high-density peptide array
with 2.1 million peptides.
• Peptides composed of 12AA with
6AAs overlap.
Results
• IgG was screened for in 57 serum
samples.
• Of the 11520 antigens found,
10,846 were unique.
• Female narcolepsy patients had
higher reactivity compared to male
patients (p=0.004).
• The final selection was 14 antigens
to show disease related reactivity.
Results Cont.
• 14 antigens were reactivated for
second verification.
• Used for the sample set from
Sweden.
• METTL22 and NT5C1A were
verified to show higher reactivity in
the Swedish patients.
• METTL22= 23% reactivated
• NT5C1A= 21% reactivated
Further Investigation of NT5C1A
• Showed reactivity in all sample
groups but not a higher frequency
for narcolepsy.
• Tested with patients with inclusion
body myositis.
• Statistical significance only the Cterminal antigen.
Discussion
• 4 of the 14 proteins revealed
sequence similarity when compared
to the H1N1 virus strain used in
the vaccine.
• GLS2 sequence is an enzyme that
hydrolyses glutamine to glutamate.
Important for neurophysiological and
homeostasis functions.
H1N1 influenza virus induces
narcolepsy-like sleep disruption
and targets sleep-wake regulatory
neurons in mice.
Chiara Tesoriero et. al.
Or, Mr. Bean goes on
vacation, contracts
H1N1, and gets a
souvenir called
narcolepsy.
Materials and Methods
• Animals: Female K/O mice for Rag1-/-, and wild-type mice.
• Influenza A/WSN/33
• Surgery: Implantation of wireless NeuroLogger microchips.
• Real-Time PCR: With custom designed primers on an ABI
Prism 7000 real-time thermocycler.
Female K/O mice for Rag1-/• Rag= Recombination activating gene
• Responsible for enzyme encoding in immunoglobulin and Tcell receptor molecules.
• Rag1 -/- have no memory T-cells and no B-cells to fight the
influenza virus.
Materials and
Methods Cont’d.
• Mice were instilled
intranasally with H1N1
influenza A virus or saline.
• Wake and sleep states were
recorded before infection
(baseline).
• Electroencephalography(E
EG) and
electromyography(EMG)
were used to measure slow
wave sleep (SWS) and
REM.
Infection
• At time 0 one group of mice (n = 13.
• 6 of the infected mice used for
EEG/EMG/actimetry recording) was
infected.
• control group was exposed to saline.
• Mice that had a 15% body loss were
killed for ethical reasons.
Fig. 1: Experimental time line and survival rate
of Rag1−/− mice following intranasal infection
with the WSN/33 strain of influenza A virus.
Results
• Sleep and wake states were analyzed at
2,3, and 4 weeks post infection.
• These were compared with baseline
readings.
• In the control Rag -/- no significant
changes were detected after saline
treatment.
First Three Weeks
• No significant change was noticed
in the total time in each state during
the light phase.
• No significant alterations were
found in the analyzed sleep and
wake patterns
Week 4: when sleep/wake cycle got weird.
• The number of state episodes and
sleep-wake transitions increased.
• Reduced mean REM sleep latency
was recorded.
• Resulting in a marked
fragmentation of sleep.
• Rapid cycling between state cycles.
Week of craziness cont.
• SOREM episodes, not seen in the
saline-treated controls appeared 4th
week post-infection.
• # of state episodes & sleep-wake
tranitions also increased in mice where
mean wake episodes decresed.
• Resulting in frequent intrusion of
sleep into wakefulness.
• SOREM episodes occurred during
dark phase of infected mice.
Spectral Power Analysis
• No differences between infected
and control found.
• For wakefulness, SWS, REM.
• Infected mice showed significant
increase in SWS theta bands.
• Similar to those observed in a
conditional ablation model of
Orx/Hcrt deficiency.
Innate-Immune Responses
• Mediators of IIR are released by
host during infection to limit viral
spread.
• TNF-alpha, IL-beta, and IFN-beta
codings increased.
• Nitric oxide synthase did not
increase—involved in SWS
recovery after sleep deprivation.
Influenza targets brain areas in SWS regulation.
• Viral antigens were seen in a few
trigeminal ganglia neurons
• These were highly expressed dorsal
trigeminal root central to
periphery-brain border.
• Virus spread from olfactory nerve
layer to deeper layers in the
olafactory bulb.
Influenza vs. Sleep-Wake Regulation cont.
• However, infection was constricted
to olfactory and trigeminal systems.
• On rare occurrances virus did spread
into the neocortex and hippocampus
Influenza vs. Sleep-Wake Regulation cont.
• Rostro-caudal spread was bilateral,
with exception of the LH.
• Orx/Hcrt neurons and neurons
containing melanin-concentrating
hormone (MCH) were infected.
• Well established involvement in sleepwake reg. Reduced number of
Orx/Hcrt neurons
Influenza vs. Sleep-Wake Regulation cont.
• Cresyl violet staining shows cell
loss in Orx/Hcrt areas.
• No cell loss where MCH neurons
are found.
• Contralateral, noninfected neurons
appear normal. (E,G)
Influenza vs. Sleep-Wake Regulation cont.
• Microglia gradually increase from
periphery to infected lesions.
Suggesting activation. (I)
• Astrocytes show features of
activation in periphery of heavily
affected area. (I)
• Viral antigens were seen within
astrocytes.
• Other infection & double staining
patterns used to detect other areas
involved in sleep-wake regulation.
• Cholinergic ventral forebrain (A, B)
• Histaminergic neurons in TMN (C,D)
• Transition zone between VTA and
overlapping with dopamine neurons (E,F)
• DRN/MRN neurons
Discussion
• 4th week post-infection was when
the observed changes in the sleepwake pattern, similar to models of
narcolepsy.
• Frequent episodes of sleep
interruption are similar to what
occur in Orx/Hcrt-deficient mice.
Discussion Cont.
• Antibodies to the NP can crossreact with human hypocretin
receptor 2, which may further
interfere with Orx/Hcrt signaling.
Discussion
Cont.
• Proposed route of the
influenza infection.
• Influenza A infections are
limited to respiratory
epithelium
• Important to determine
what controls spread into
other nervous tissue.