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More than Neurons;How Astroglia contribute to cognitive deficits of Trisomy 21
HMB420: Kathleen and Nouran
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Goals- Understand the neurodevelopmental disorder and
affected cell types- Trisomy 21; cognitive and developmental effects
- Understand the research methods- Yamanaka reprogramming method, astrocytes’ extracellular
medium, electrophysiology, microarray genomic analysis, transplantation in vivo, Minocycline and siRNA effects.
- Understand what was found- Altered growth patterns, multiple proofs of findings, possible
treatment options.
- Understand the implications of this research - Re-defining neurodevelopmental disorders? New treatments? - Pros and Cons of this paper
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● Complexity of the brain largely due to astroglial function● Astroglia
o Support neuronal survival and neurogenesis
o Support axon and dendritic growth
o Release apolipoprotein E (ApoE), thrombospondins (TSPs), SPARCL1, SPARC, and more
o Promote synaptogenesis and differentiation from Neural Progenitor Cells (NPCs)
(Clarke and Barres, 2013)
Astroglia and the Human Brain
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● Complexity of the brain largely due to astroglial function● Astroglia
o Support neuronal survival and neurogenesis
o Support axon and dendritic growth
o Release apolipoprotein E (ApoE), thrombospondins (TSPs), SPARCL1, SPARC, and more
o Promote synaptogenesis and differentiation from Neural Progenitor Cells (NPCs)
(Clarke and Barres, 2013)
Astroglia and the Human Brain
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● Complexity of the brain largely due to astroglial function● Astroglia
o Support neuronal survival and neurogenesis
o Support axon and dendritic growth
o Release apolipoprotein E (ApoE), thrombospondins (TSPs), SPARCL1, SPARC, and more
o Promote synaptogenesis and differentiation from Neural Progenitor Cells (NPCs)
(Clarke and Barres, 2013)
Astroglia and the Human Brain
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Composition of the brain
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Down Syndrome - Genetics● Nondisjunction or Robertsonian
translocation● Altered gene expression profile;
APP Amyloid precursor protein - Cognitive difficulties
SOD1 Superoxide dismutase - Anti-oxid ant & role in Alzheimer's
DSCR1 Down Syndrome Critical Region Gene 1 - Signal transduction
DYRK Dual-specificity Tyrosine Phosphorylation-Regulated Kinase - Neurogenesis
IFNAR Interferon, Alpha, Beta, and Omega, Receptor - Immune / cognitive
ETS2 Avian Erythroblastosis Virus E26 Oncogene Homolog 2 - Apoptosis
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Down Syndrome - Genetics● Nondisjunction or Robertsonian
translocation● Altered gene expression profile;
APP Amyloid precursor protein - Cognitive difficulties
SOD1 Superoxide dismutase - Anti-oxid ant & role in Alzheimer's
DSCR1 Down Syndrome Critical Region Gene 1 - Signal transduction
DYRK Dual-specificity Tyrosine Phosphorylation-Regulated Kinase - Neurogenesis
IFNAR Interferon, Alpha, Beta, and Omega, Receptor - Immune / cognitive
ETS2 Avian Erythroblastosis Virus E26 Oncogene Homolog 2 - Apoptosis
Wild-type
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Down Syndrome - Genetics● Nondisjunction or Robertsonian
translocation● Altered gene expression profile;
APP Amyloid precursor protein - Cognitive difficulties
SOD1 Superoxide dismutase - Anti-oxid ant & role in Alzheimer's
DSCR1 Down Syndrome Critical Region Gene 1 - Signal transduction
DYRK Dual-specificity Tyrosine Phosphorylation-Regulated Kinase - Neurogenesis
IFNAR Interferon, Alpha, Beta, and Omega, Receptor - Immune / cognitive
ETS2 Avian Erythroblastosis Virus E26 Oncogene Homolog 2 - Apoptosis
Trisomy 21
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Down Syndrome - Genetics● Nondisjunction or Robertsonian
translocation● Altered gene expression profile;
APP Amyloid precursor protein - Cognitive difficulties
SOD1 Superoxide dismutase - Anti-oxid ant & role in Alzheimer's
DSCR1 Down Syndrome Critical Region Gene 1 - Signal transduction
DYRK Dual-specificity Tyrosine Phosphorylation-Regulated Kinase - Neurogenesis
IFNAR Interferon, Alpha, Beta, and Omega, Receptor - Immune / cognitive
ETS2 Avian Erythroblastosis Virus E26 Oncogene Homolog 2 - Apoptosis
Robertsonian Translocation
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Down Syndrome - Genetics● Nondisjunction or Robertsonian
translocation● Altered gene expression profile;
APP Amyloid precursor protein - Cognitive difficulties
SOD1 Superoxide dismutase - Anti-oxid ant & role in Alzheimer's
DSCR1 Down Syndrome Critical Region Gene 1 - Signal transduction
DYRK Dual-specificity Tyrosine Phosphorylation-Regulated Kinase - Neurogenesis
IFNAR Interferon, Alpha, Beta, and Omega, Receptor - Immune / cognitive
ETS2 Avian Erythroblastosis Virus E26 Oncogene Homolog 2 - Apoptosis
Possible Gametes
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Down Syndrome● Many brain abnormalities in Trisomy 21
including: - Reduced brain volume- Impaired neurogenesis- Abnormal synaptic and
dendritic morphology
● Reduced cell number and volume of the Dentate Gyrus, Hippocampus, Parahippocampal Gyrus
● Reduced proliferation in Hip and the PHG● Increased cell death in Hip
Guidi et al. (2007):
Alzheimer's diseaseAmyotrophic lateral sclerosisAutoimmune polyendocrine syndromeDown syndromeErondu–Cymet syndromeHolocarboxylase synthetase deficiencyHomocystinuriaJervell Lange-Nielsen syndromeLeukocyte adhesion deficiencyMajewski osteodysplastic dwarfism type II Romano-Ward syndrome
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● No good developmental model to study DS● Limited by DS fetal tissue attainability● Murine models only partial triplication of Ch21
● Brain complexity differences by astroglial cells
Previous studies have solely focused on the neuronal role in DS brain abnormalities, while the astroglial role
has been largely neglected
Reason for Experimentation
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Role of Astroglial in DS
Induced pluripotent stem cells (iPSC) to:Investigate how astroglia contribute to pathogenesis of DS and
explore potential therapeutic treatment for DS
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Experimental Overview1. Create human induced Pluripotent Stem Cells (hiPSC)2. Observe development differences in DS and control neurons /
astrocytes3. Observe effects of DS and control astroglia on neurons and NPCs4. Observe DS and control astroglia induced neuronal development5. Observe gene expression profiles of DS and control astroglia6. Observe effects in vivo7. Observe effects of Minocycline treatment on DS astroglia
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● Yamanaka reprograming○ Transfected with 4 µg of moloney murine leukemia-
based retroviral vectors ○ cDNA of OCT4, SOX2, KLF4 / c-MYC
■ Lipofectamine ○ 1.2 X 105–2 X 105 DS fibroblasts onto each well○ Neural rosettes => Neurospheres
● Karyotyping & In situ fluorescence○ 2 DS NPC lines
Inducing Stem Cells
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Why Minocycline?● Anti-inflammatory antibiotic drug that is used in
the treatment of infections ● Neuroprotective effects
○ Ischemic Injury○ Parkinson’s Disease○ Huntington’s Disease
(Plane et al., 2010)
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DS Astroglia and NeurogenesisExamined the neuronal differentiation in NPCs - Incubated with differentiating factors- Incubated with no differentiating factors
Assessed:● % of ßTubulin III + neurons● % of S100B + astroglia● Neurite development
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DS hiPSC Morphology● hiPSC differentiation and
development○ DS neurons normal if differentiated○ if not induced; DS NPCs preferentially
develop into astroglia ○ if not induced; DS neurons have
reduced neurite development
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Survival; DS Astroglia ACM● Maintained DS and control astroglia in astroglial-
cell conditioned medium (ACM) ○ Assessed nitrite/nitrate concentrations
● DS and control astroglia○ Reactive oxygen species (ROS) and induced nitric oxide
synthase (iNOS)○ Proliferation rate ○ Glutamate (Glu) uptake
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Survival; DS Astroglia ACM● Striking differences
between DS and Control astroglia○ More iNOS / Nitrate○ More ROS in DS○ More proliferation in DS○ More Glut uptake in DS
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Survival; DS Astroglia ACM● Striking differences between
DS and Control astroglia○ More cell death/ caspase
activation ○ Reduced neurite development
● DS and Control rescue■ siRNA/ Minocycline
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Development; DS ACM● DS neurons in:
- DS ACM- Control ACM- DS siRNA ACM- DS + minocycline ACM
● Neurophysiological assessment
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Development; DS ACM● Altered electrophysiology;
○ Resting membrane potential○ Resistance ○ Capasatance○ sEPSP hz & strength○ Na current○ vgK current○ Rectifying K current
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DS Astroglial Gene Analysis ● Gene expression profiles
○ S100B, GFAP, NFE2L2 and TSPs○ Neurotrophic factors (BDNF, BMP)
● Minocycline Treatment ○ Assessed differences in gene profiles in DS
Astroglia ○ Assessed phenotypic differences in DS Astroglia
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DS Astroglial Gene Analysis ● Altered gene expression● Similar to experiments
○ NFE2L2○ Oxidative stress○ Synaptogenesis○ Maturation○ BMP & FGF
● Rescued by minocycline
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DS Astroglia Di/Tri HSA21 ● Isolated isogenic disomic and trisomic iPSCs
from patient DS-3○ Di-DS3○ Tri-DS3
● Differentiated Di-DS3 and Tri-DS3 → Astroglia● Compared phenotypic differences between Tri-
DS3 astroglia, Di-DS3 and control astroglia
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DS Astroglia Di/Tri HSA21 Compared to Di-DS3:● Tri-DS3 → higher expression of S100B,
GFAP, iNOS, lower expression of TSP-1 and TSP-2○ Minocycline treatment → corrected
gene expression● Tri-DS3 neurons spontaneously
differentiated showed decreased neurite length
● Tri-DS3 ACM caused more DS neuronal cell death ○ Tri-DS3-Minco → reduced DS
neuronal cell death
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DS Astroglia in vivo Transplantation Study
Di-DS3 and Tri-DS3 astroglia were transplanted into the lateral ventricles (LV) of immunodeficient mice (rag1 -/-)
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DS Astroglia in vivo ● Integration and survival of
the di/tri-DS3 astroglia in immunodeficient mouse brains
● Di-DS3 astroglia promoted neurogenesis and increased proliferation in dorsal SVZ, Tri-DS3 astroglia did not
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Discussion● Found;
○ Altered morphology in neurons due to DS astrocytes○ Altered cell death in neurons due to DS astrocytes○ Altered electrophysiology due to DS astrocytes○ Altered genomic expression in DS astrocytes
explaining observed effects■ proven by Di-DS2/ Tri-DS2
○ Altered activity in DS astrocytes in vivo confirming observed effects
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Importance● Huge implications for many neuronal diseases!
○ Possible treatment strategies to rescue neuronal development / degeneration, not just in DS
● Re-thinking developmental disorders as ‘neuron-based’○ Exploring the contribution of glial cells to the pathophysiology of
diseases
● One of the first studies to elucidate the role of astroglial function in the abnormal neurobiology of Down Syndrome and investigating them as potential therapeutic targets for drug treatment
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Future Work● Studying the DS and new potential
treatments in vivo ○ ‘Humanized mouse models’
● Examining the role of astroglia in neurodegenerative diseases such as Alzheimer’s Disease
● Further explore the effects of minocycline on DS neurobiology in human clinical trials
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Criticisms● Potential skewing of results due to individual differences
among subjects ○ e.g. DS1 showing > 74-fold increase in S100B gene expression
compared to approximately 2.5 fold in DS2● Focused on what was agreed to hypothesis
What about gene expression?Not pointing out deviance in gene expression results
● Different types of astroglia - variation○ More needs to be said about the specific contribution of each type of
astroglial cell
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Any Questions?
References
1. Guidi, S. et al. Neurogenesis impairment and increased cell death reduce total neuron number in the hippocampal region of fetuses with Down syndrome. Brain Pathol. 18, 180–197 (2008).
2. Chen, C. et al. Role of astroglia in Down’s syndrome revealed by patient-derived human-induced pluripotent stem cells. Nat Commun. 5, 1-18 (2014).