This document summarizes a study that investigated how astroglia contribute to cognitive deficits in Down syndrome (DS) using human induced pluripotent stem cell technology. The study found that DS astrocytes exhibited altered gene expression, increased oxidative stress, and promoted less neuronal development and survival compared to controls. Treating DS astrocytes with minocycline reduced these effects by correcting gene expression. The findings suggest astroglia play an underappreciated role in DS pathology and represent a potential therapeutic target.

1. More than Neurons;
How Astroglia contribute to cognitive deficits of Trisomy 21
HMB420: Kathleen and Nouran

2. 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

3. ● 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

4. ● 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

5. ● 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

6. Composition of the brain

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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 disease
Amyotrophic lateral sclerosis
Autoimmune polyendocrine
syndrome
Down syndrome
Erondu–Cymet syndrome
Holocarboxylase synthetase
deficiency
Homocystinuria
Jervell Lange-Nielsen syndrome
Leukocyte adhesion deficiency
Majewski osteodysplastic
dwarfism type II
Romano-Ward syndrome

13. ● 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

14. 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

15. Experimental Overview
1. Create human induced Pluripotent Stem Cells (hiPSC)
2. Observe development differences in DS and control neurons /
astrocytes
3. Observe effects of DS and control astroglia on neurons and NPCs
4. Observe DS and control astroglia induced neuronal development
5. Observe gene expression profiles of DS and control astroglia
6. Observe effects in vivo
7. Observe effects of Minocycline treatment on DS astroglia

16. ● 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

17. 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)

18. DS Astroglia and Neurogenesis
Examined the neuronal differentiation in NPCs
- Incubated with differentiating factors
- Incubated with no differentiating factors
Assessed:
● % of ßTubulin III + neurons
● % of S100B + astroglia
● Neurite development

19. 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

20. 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

21. 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

22. 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

23. Development; DS ACM
● DS neurons in:
- DS ACM
- Control ACM
- DS siRNA ACM
- DS + minocycline ACM
● Neurophysiological assessment

24. Development; DS ACM
● Altered electrophysiology;
○ Resting membrane potential
○ Resistance
○ Capasatance
○ sEPSP hz & strength
○ Na current
○ vgK current
○ Rectifying K current

25. 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

26. DS Astroglial Gene Analysis
● Altered gene expression
● Similar to experiments
○ NFE2L2
○ Oxidative stress
○ Synaptogenesis
○ Maturation
○ BMP & FGF
● Rescued by minocycline

27. 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

28. 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

29. DS Astroglia in vivo
Transplantation Study
Di-DS3 and Tri-DS3 astroglia
were transplanted into the
lateral ventricles (LV) of
immunodeficient mice
(rag1 -/-)

30. 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

31. 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

32. 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

33. 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

34. 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

35. 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).