Neurodegeneration

2. Introduction
ApoE
 Class of apolipoprotein, mediates cholesterol metabolism,
 In the brain, mainly produced by astrocytes, as the principal
cholesterol carrier to neuron via ApoE receptor.
 Three polymorphic APOE alleles in the human population (APOE2,
APOE3, and APOE4)
 APOE4 has a frequency of 14% in general, strong genetic risk
factor for Alzheimer’s disease
 Apo-E affects amyloid-β (Aβ) clearance, aggregation and
deposition in an isoform-dependent manner
 Human ApoE increased APP transcription and Aβ production

3. Introduction
 Correlation of ApoE protein with amyloid-β

4. Introduction
Tau protein
 A kind of microtubule-associated protein(MAP)
 6 isoform by alternative splicing
 Intrinsically disordered in normal condition
 Phosphorylated by many kinds of kinase such as Cdk5, PKA, MAPK, etc.
 Tau mutation (P301S, S305N, V337M, K369I, R406W...) is found in
frontotemporal dementia with parkinsonism linked to chromosome
17(FTDP-17) patients
 Insoluble-hyperphosphorylated tau forms neurofibrillary tangle(NFT) in
the developed pathological condition.
 Recently reported about neuro toxicity of soluble oligomeric tau
 NFT appeared in entorhinal cortex firstly, tau aggaregation distributed
other region.

5. Introduction
 6 isoforms of tau protein
Mutations found in FTDP-17

6. Introduction
 Revealed correlation of ApoE protein with
tau protein

7. Materials & Methods
 Animals
 Human ApoE2, ApoE3, and ApoE4 KI mice (C57BL/6),ApoE KO mice (C57BL/6) (The Jacksonlaboratory, 002052)
->E2, E3, E4 mouse
 P301ShE/hE: P301S tau transgenic mice (C57BL/6, The Jackson Laboratory, 008169) -expressing human P301S
1N4R tau- crossed to human ApoE KI mice for all three ApoE isoforms
->TE2, TE3, TE4 mouse
 P301ShE/− mice: P301ShE/hE crossed to ApoE KO mice
 P301S−/− for all three ApoE isoforms (P301ShE/− mice were crossed with hE/− mice)
->TEKO (combined all ApoE isoforms)
 Immunohistochemistry
P-tau, AT8
CD68, GFAP
 Volumetric analysis
Sudan black staining – lipid or lipoprotein
Every sixth coronal brain section (300um between sections)
volume = (sum of area) × 0.3 mm
started at bregma − 2.3 mm and ended at bregma − 3.9 mm
 ELISA
Human tau and ApoE, cytokines
 Western blot
GFAP, GAPDH, ApoE, α tubulin
 qRT-PCR

8. Materials & Methods
 Neuronal layer thickness measurement
Three sections (bregma − 1.4, − 1.7, and − 2.0 mm) from each mouse
Cresyl violet staining
The thicknesses of the CA1 pyramidal cell layer and dentate gyrus granular cell layer
 Brain extraction- sequential biochemical extraction
For separately detecting soluble tau and insoluble tau.
with RAB (aqueous buffer), RIPA (radio immunoprecipitation assay, detergent buffer),
and FA (70% formic acid)
 Recombinant ApoE treatment on primary cultured neuron
 Glia-neuron co-culture
 LPS stimulation on primary cultured microglia and cytokine measurement
 Study of neurodegeneration in human primary tauopathies
 Analysis of clinical disease progression in human Alzheimer disease patients

9. Fig. 1. Regulating tau-mediated neurodegeneration by ApoE
Extended Fig.1.
9-month-old TE mouse
3-month-old TE mouse
9-month-old non-tau transgenic mouse
 Brain volume measurement
 ApoE regulates tau-mediated
neurodegeneration getting severer.
 ApoE4 exacerbate neurodegeneration in
tauopathy progress.

10. Fig. 1. Regulating tau-mediated neurodegeneration by ApoE
 Neuronal layer thickness
Granule cell layer and dentate gyrus
CA1 region pyramidal layer
Extended Fig.2.
 ApoE regulates tau-mediated neurodegeneration
getting severer.
 ApoE4 exacerbate neurodegeneration in tauopathy
progress.

11. Fig. 2. ApoE genotypes differentially regulate tau pathology.
 Quantitative ELISA for tau detection after sequential biochemical brain extraction
Extended Fig.3.
Extended Fig.3.
 ApoE4 affect increasing soluble tau at the onset of the disease,
insoluble tau in developed tau pathology condition.
 ApoE4 affect autophagy-mediated tau clearance.

12. Fig. 2. ApoE genotypes differentially regulate tau pathology.
 Hyperphosphorylation of tau can be elevated by ApoE4.
 Pathological p-tau can be distributed from axon to cell
body in dentate gyrus granule cells in hippocampus.
 Type 4 p-tau pattern-correlated severe atrophy- is highly
detected in TE4 mouse.

13. Fig. 3. ApoE strongly modulates microglial activation
 ApoE4 has higher immune reactivity than other isoforms.
 Proinflammatory upregulated genes increased by ApoE4.

14. Fig. 3. ApoE strongly modulates microglial activation
CD68 signal upregulated in 9-month-old TE4
 ApoE4 induces high immune reactivity and
neuroinflammation.
Extended Fig.6.

15. Fig. 4. Astrocytic activation and increased neuronal death by ApoE4 in vitro
GFAP signal upregulated 9-month-old TE4
 ApoE4 activated A1-specific (activated in only inflammatory condition), not A2-specific gene.
 ApoE4 regulates tau inducing neuroinflammation.

16. Fig. 4. Astrocytic activation and increased neuronal death by ApoE4 in vitro
Acctivated astrocytes play an important role in inducing neuronal death.
ApoE itself was directly involved in inducing neurotoxicity in P301S tau-expressing susceptible neurons

17. Conclusion