The Role of Astroglia in the Development of Down Syndrome

More than Neurons;How Astroglia contribute to cognitive deficits of Trisomy 21

HMB420: Kathleen and Nouran

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

● 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

Composition of the brain

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

Trisomy 21

Robertsonian Translocation

Possible Gametes

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

● 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

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

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

● 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

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)

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

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

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

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

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

Development; DS ACM● DS neurons in:

- DS ACM- Control ACM- DS siRNA ACM- DS + minocycline ACM

● Neurophysiological assessment

Development; DS ACM● Altered electrophysiology;

○ Resting membrane potential○ Resistance ○ Capasatance○ sEPSP hz & strength○ Na current○ vgK current○ Rectifying K current

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

DS Astroglial Gene Analysis ● Altered gene expression● Similar to experiments

○ NFE2L2○ Oxidative stress○ Synaptogenesis○ Maturation○ BMP & FGF

● Rescued by minocycline

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

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

DS Astroglia in vivo Transplantation Study

Di-DS3 and Tri-DS3 astroglia were transplanted into the lateral ventricles (LV) of immunodeficient mice (rag1 -/-)

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

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

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

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

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

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).

The Role of Astroglia in the Development of Down Syndrome

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