Cytokines analysis in therapeutic and sub-therapeutic lithium-treated primary neuron and glia...
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a test based on peripheral blood leukocyte infection by vesicular stomatitis virus(VSV). Results: The resistance to VSV infection was individually dif- ferentiated and depended on donor age. The strongest antiviral resistance was observed in 30-40 year olds and the weakest in the group over 60 years. Conclusions: The decline of VSV resistance is observed in both non-clin- ical as well as in clinical (cancer and AD) samples over 60. The possible stimulation of leukocyte resistance by plant-made or synthetic drugs is dis- cussed. The microglia and neuronal cells are directly involved in inflamma- tory process in Alzheimer’s disease. Probably the long-term activation of the innate immune system leads to an inflammatory reaction that coverges in cy- tockeletal alterations such as tau aggregation and paired filament formation and accelerates neuronal cell death.Search for novel drugs that can attenuate inflammatory and autoimmune reactions and improve cognitive autcomes of Alzheimer patients remain important goals for the near future. P2-018 MICROGLIA CONSTITUTE A BARRIER THAT PREVENTS THE FORMATION OF NEUROTOXIC OLIGOMERIC BETA-AMYLOID HOT SPOTS AROUND PLAQUES Carlo Condello 1 , Peng Yuan 2 , Pritam Das 3 , Jaime Grutzendler 2 , 1 University of California San Francisco, San Francisco, California, United States; 2 Yale University, New Haven, Connecticut, United States; 3 Mayo Clinic Florida, Jacksonville, Florida, United States. Contact e-mail: [email protected] Background: In Alzheimer’s disease (AD), microglia processes are fre- quently found wrapping around the fibrillar amyloid plaque core which is generally surrounded by a halo of neurotoxic b-amyloid (Ab) oligomers. It is not known however, whether microglia affect the dynamic equilibrium between soluble oligomeric Ab and amyloid plaque fibrils, and thus insulate neurons from the accumulation of toxic Ab aggregates. Given that microglia can produce a number of molecules with potential neurotoxic effects, it has frequently been assumed that their activation around Ab deposits is part of a pathological reaction that causes harm to adjacent neurons. However, anti- inflammatory strategies to treat AD and other neurodegenerative conditions have so far been unsuccessful. This raises the possibility that microglia play important protective functions which may be abolished by their inhibition. Methods: To determine whether microglia can limit Ab aggregation and toxicity in vivo, we designed novel methods to study the interactions be- tween amyloid plaques, soluble Ab, microglia and neurons in the living brains of transgenic AD mouse models using two-photon imaging and in fixed brain slices with high-resolution confocal microscopy. Results: Sub- arachnoid infusion of soluble oligomeric b-amyloid (Ab) demonstrated rapid and specific binding to preexisting plaques. Interestingly, discrete hot-spots of high-affinity Ab binding were found within the plaque halo and coincided with areas of low fibrillar Ab density. Surprisingly, in early stages of plaque formation, the hot-spot location was strongly anti-corre- lated with microglia processes suggesting these cells act as a barrier that limits outward plaque expansion. Neurites in the vicinity of microglia-un- covered areas were exposed to the Ab hot-spot leading to prominent injury. In aging, microglia coverage was dramatically reduced, resulting in en- larged areas of the Ab halo and dystrophic neurites. Strikingly, when micro- glia activity was stimulated by passive immunization using the Ab antibody, AB9, or by AAV-mediated interleukin-6 overexpression the barrier effect was greatly enhanced leading to reduced neurotoxicity. Conclusions: Thus in early stages of AD, microglia play a critical barrier role to restrict plaque expansion and insulate neurons from neurotoxic Ab hot spots, a mechanism that could be exploited therapeutically. P2-019 CYTOKINES ANALYSIS IN THERAPEUTIC AND SUB-THERAPEUTIC LITHIUM-TREATED PRIMARY NEURON AND GLIA CO-CULTURE Daniel Kerr 1 , Vanessa De Paula 2 , Leda Talib 3 , Wagner Gattaz 4 , Orestes Forlenza 5 , 1 LIM-27, S~ ao Paulo, Brazil; 2 Laboratory of Neuroscience - LIM 27, Department and Institute of Psychiatry, Faculty of Medicine, Un, S~ ao Paulo, Brazil; 3 Laboratory of Neuroscience (LIM-27), Department and Institute of Psychiatry, Faculty of Medicine, Un, S~ ao Paulo, Brazil; 4 USP, S~ ao Paulo, Brazil; 5 University of S~ ao Paulo, S~ ao Paulo - S.P., Brazil. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD), supposed to affect 35 million peo- ple around the world, is the most common cause of dementia. The biochem- ical hallmarks of AD are the senile plaques (highly insoluble amyloid beta peptide deposits) and neurofibrillary tangles. Those lead to damaged neu- rons and neurites and provide obvious stimuli for inflammation. Despite im- portant mechanisms have been discovered, there is no effective treatment for AD yet. Recent publications suggest lithium as a possible treatment for AD, not only for its progression, but also prevention. Also, lithium has been shown to act as a positive modulator of anti-inflammatory cascades in neu- rons cell and animal models. Here we investigate the effect of low doses of lithium in several pro and anti-inflammatory cytokines in a primary hippo- campal or cortical neuron and glia co-culture model. Methods: Primary hip- pocampal and cortical neurons or glia cells of Wistar rats embryos were cultured separately. After 2 days in culture (DIC) the glia cells inserts were added to the neuronal cell cultures. At 4 DIC we added to the culture medium different concentrations of lithium chloride (0, 0.02, 0.2, 2mM). At 10 DIC culture medium was collected and 10 cytokines (GM-CSF, IN- Fgamma, IL-10, IL-12, IL-1beta, IL-4, IL-5, TNFalpha, IL-2 and IL-6) con- tent was evaluated with a multiplex Luminex assay. Results: IL-2 and 6 were not detected by our assay. The other cytokines values are summarized in Table1. All lithium treated groups were compared to the respective un- treated control. Statistical significance was assessed with Student t Test. Conclusions: Lithium treatment changed cytokines secretion levels in our co-culture model. Both anti and pro-inflammatory cytokines significantly increased in cortex and hippocampus co-culture. Anti-inflammatory cyto- kines IL-5 and GM-CSF significantly decreased in cortex co-culture. There is no clear tendency of change in the studied cytokines, however, it is impor- tant to keep in mind that in our model there is no external inflammatory agent and the changes observed are due to the physiological effect of lithium alone. P2-020 MICRORNA COMPLEXITY IN ALZHEIMER’S DISEASE CEREBROSPINAL FLUID, EXTRACELLULAR FLUID AND BRAIN TISSUE BIOPSY Walter Lukiw 1 , Prerna Dua 2 , JM Hill 3 , S Bhattacharjee 3 , Yuhai Zhao 4 , Hilary Thompson 5 , 1 Louisiana State University Neuroscience Center, New Orleans, Louisiana, United States; 2 Louisiana Technical University, Ruston, Louisiana, United States; 3 Louisiana State University, New Table 1 The effect of lithium in cytokines in the culture medium of primary hippocampal and cortical neuron and glia co-culture. Values presented as percentage of control (100%). The values of those with an average similar to control were omited. * p<0.05 in Student’s t-Test. Lithium Cortex Hippocampus 0.02mM 0.2mM 2mM 0.02mM 0.2mM 2mM Pro-inflammatory IL-1Beta 104* 104 106 102 103* IL-12 103* 107 98 98 TNF-alpha 103* 104* 108* 102* 101 Anti-inflammatory IL-4 107* 105 105 IL-5 93* 92 91 105* 105 IL-10 108* 138 122* 147 INF-gamma 109* 114 106* 112 GM-CSF 93 87* 90 120* 77 121 Monday, July 15, 2013: Poster Presentations: P2 P351
Transcript of Cytokines analysis in therapeutic and sub-therapeutic lithium-treated primary neuron and glia...
Monday, July 15, 2013: Poster Presentations: P2 P351
a test based on peripheral blood leukocyte infection by vesicular stomatitis
virus(VSV). Results: The resistance to VSV infection was individually dif-
ferentiated and depended on donor age. The strongest antiviral resistance
was observed in 30-40 year olds and the weakest in the group over 60 years.
Conclusions: The decline of VSV resistance is observed in both non-clin-
ical as well as in clinical (cancer and AD) samples over 60. The possible
stimulation of leukocyte resistance by plant-made or synthetic drugs is dis-
cussed. The microglia and neuronal cells are directly involved in inflamma-
tory process in Alzheimer’s disease. Probably the long-term activation of the
innate immune system leads to an inflammatory reaction that coverges in cy-
tockeletal alterations such as tau aggregation and paired filament formation
and accelerates neuronal cell death.Search for novel drugs that can attenuate
inflammatory and autoimmune reactions and improve cognitive autcomes of
Alzheimer patients remain important goals for the near future.
P2-018 MICROGLIA CONSTITUTE A BARRIER THAT
PREVENTS THE FORMATION OF NEUROTOXIC
OLIGOMERIC BETA-AMYLOID HOT SPOTS
AROUND PLAQUES
Table 1
The effect of lithium in cytokines in the culture medium of primary
hippocampal and cortical neuron and glia co-culture. Values presented as
percentage of control (100%). The values of thosewith an average similar to
control were omited. * p<0.05 in Student’s t-Test.
Lithium
Cortex Hippocampus
0.02mM 0.2mM 2mM 0.02mM 0.2mM 2mM
Pro-inflammatory
IL-1Beta 104* 104 106 102 103*
IL-12 103* 107 98 98
TNF-alpha 103* 104* 108* 102* 101
Anti-inflammatory
IL-4 107* 105 105
IL-5 93* 92 91 105* 105
IL-10 108* 138 122* 147
INF-gamma 109* 114 106* 112
GM-CSF 93 87* 90 120* 77 121
Carlo Condello1, Peng Yuan2, Pritam Das3, Jaime Grutzendler2,1University of California San Francisco, San Francisco, California, United
States; 2Yale University, New Haven, Connecticut, United States; 3Mayo
Clinic Florida, Jacksonville, Florida, United States.
Contact e-mail: [email protected]
Background: In Alzheimer’s disease (AD), microglia processes are fre-
quently found wrapping around the fibrillar amyloid plaque core which is
generally surrounded by a halo of neurotoxic b-amyloid (Ab) oligomers.
It is not known however, whether microglia affect the dynamic equilibrium
between soluble oligomeric Ab and amyloid plaque fibrils, and thus insulate
neurons from the accumulation of toxic Ab aggregates. Given that microglia
can produce a number of molecules with potential neurotoxic effects, it has
frequently been assumed that their activation around Ab deposits is part of
a pathological reaction that causes harm to adjacent neurons. However, anti-
inflammatory strategies to treat AD and other neurodegenerative conditions
have so far been unsuccessful. This raises the possibility that microglia play
important protective functions which may be abolished by their inhibition.
Methods: To determine whether microglia can limit Ab aggregation and
toxicity in vivo, we designed novel methods to study the interactions be-
tween amyloid plaques, soluble Ab, microglia and neurons in the living
brains of transgenic AD mouse models using two-photon imaging and in
fixed brain slices with high-resolution confocal microscopy. Results: Sub-
arachnoid infusion of soluble oligomeric b-amyloid (Ab) demonstrated
rapid and specific binding to preexisting plaques. Interestingly, discrete
hot-spots of high-affinity Ab binding were found within the plaque halo
and coincided with areas of low fibrillar Ab density. Surprisingly, in early
stages of plaque formation, the hot-spot location was strongly anti-corre-
lated with microglia processes suggesting these cells act as a barrier that
limits outward plaque expansion. Neurites in the vicinity of microglia-un-
covered areas were exposed to the Ab hot-spot leading to prominent injury.
In aging, microglia coverage was dramatically reduced, resulting in en-
larged areas of the Ab halo and dystrophic neurites. Strikingly, when micro-
glia activity was stimulated by passive immunization using the Ab antibody,
AB9, or by AAV-mediated interleukin-6 overexpression the barrier effect
was greatly enhanced leading to reduced neurotoxicity. Conclusions:
Thus in early stages of AD, microglia play a critical barrier role to restrict
plaque expansion and insulate neurons from neurotoxic Ab hot spots,
a mechanism that could be exploited therapeutically.
P2-019 CYTOKINES ANALYSIS IN THERAPEUTIC AND
SUB-THERAPEUTIC LITHIUM-TREATED
PRIMARY NEURON AND GLIA CO-CULTURE
Daniel Kerr1, Vanessa De Paula2, Leda Talib3, Wagner Gattaz4,
Orestes Forlenza5, 1LIM-27, S~ao Paulo, Brazil; 2Laboratory of
Neuroscience - LIM 27, Department and Institute of Psychiatry, Faculty of
Medicine, Un, S~ao Paulo, Brazil; 3Laboratory of Neuroscience (LIM-27),
Department and Institute of Psychiatry, Faculty of Medicine, Un, S~ao Paulo,
Brazil; 4USP, S~ao Paulo, Brazil; 5University of S~ao Paulo, S~ao Paulo - S.P.,
Brazil. Contact e-mail: [email protected]
Background:Alzheimer’s disease (AD), supposed to affect 35 million peo-
ple around the world, is the most common cause of dementia. The biochem-
ical hallmarks of AD are the senile plaques (highly insoluble amyloid beta
peptide deposits) and neurofibrillary tangles. Those lead to damaged neu-
rons and neurites and provide obvious stimuli for inflammation. Despite im-
portant mechanisms have been discovered, there is no effective treatment for
AD yet. Recent publications suggest lithium as a possible treatment for AD,
not only for its progression, but also prevention. Also, lithium has been
shown to act as a positive modulator of anti-inflammatory cascades in neu-
rons cell and animal models. Here we investigate the effect of low doses of
lithium in several pro and anti-inflammatory cytokines in a primary hippo-
campal or cortical neuron and glia co-culturemodel.Methods: Primary hip-
pocampal and cortical neurons or glia cells of Wistar rats embryos were
cultured separately. After 2 days in culture (DIC) the glia cells inserts
were added to the neuronal cell cultures. At 4 DIC we added to the culture
medium different concentrations of lithium chloride (0, 0.02, 0.2, 2mM). At
10 DIC culture medium was collected and 10 cytokines (GM-CSF, IN-
Fgamma, IL-10, IL-12, IL-1beta, IL-4, IL-5, TNFalpha, IL-2 and IL-6) con-
tent was evaluated with a multiplex Luminex assay. Results: IL-2 and 6
were not detected by our assay. The other cytokines values are summarized
in Table1. All lithium treated groups were compared to the respective un-
treated control. Statistical significance was assessed with Student t Test.
Conclusions: Lithium treatment changed cytokines secretion levels in our
co-culture model. Both anti and pro-inflammatory cytokines significantly
increased in cortex and hippocampus co-culture. Anti-inflammatory cyto-
kines IL-5 and GM-CSF significantly decreased in cortex co-culture. There
is no clear tendency of change in the studied cytokines, however, it is impor-
tant to keep in mind that in our model there is no external inflammatory
agent and the changes observed are due to the physiological effect of lithium
alone.
P2-020 MICRORNA COMPLEXITY IN ALZHEIMER’S
DISEASE CEREBROSPINAL FLUID,
EXTRACELLULAR FLUID AND BRAIN TISSUE
BIOPSY
Walter Lukiw1, Prerna Dua2, JM Hill3, S Bhattacharjee3, Yuhai Zhao4,
Hilary Thompson5, 1Louisiana State University Neuroscience Center, New
Orleans, Louisiana, United States; 2Louisiana Technical University,
Ruston, Louisiana, United States; 3Louisiana State University, New
