investigating the profession of early locus coeruleus pathology in a transgenic mouse model of...
TRANSCRIPT
Mo$va$on and Background • Alzheimer’s disease (AD) is the most common
neurodegenera0ve disease and induces problems with memory, cogni0ve thinking, and behavior.
• The exact mechanisms underlying AD progression are s0ll unknown, but ß-‐amyloid (Aß) plaques and tau neurofibrillary tangles (NFT) have been implicated in the origins of AD-‐like pathology.
• Recent studies suggest that the earliest AD-‐like pathology seen in individuals lacking AD symptoms is hyperphosphorylated tau located in the locus coeruleus (LC)1.
• Made by Yoshiyama et al., the P301S-‐Tau transgenic (Tg) mouse has a muta0on that changes proline to serine at the 301st codon, which leads to hyperphosphoryla0on of tau.
• This is a model of hyperphosphorylated tau. • Studies have shown that there is
hyperphosphorylated tau in the hippocampus at six mo. in the P301S Tg mouse model, but none have inves0gated tau in the LC at earlier 0me points2,3.
Inves$ga$ng the progression of early locus coeruleus pathology in a transgenic mouse model of hyperphosphorylated tau
Aim To determine which forms of abnormal tau—hyperphosphorylated or even misfolded—are formed and when they first appear in the LC of P301S-‐Tau Tg mice, which endogenously expresses mutant human tau (hTau). I am looking at four 0me points: 2, 3, 4, and 5 months of age.
Materials and Methods P301S-‐Tau Tg Mouse • Male P301S-‐Tau Tg mice. Transcardial Perfusions and Slicing • Mice perfused at 2, 3, 4, and 5 months of age. • Drop-‐fixed in 4% paraformaldehyde. • Cryoprotected in 30% sucrose. • Sliced at 30 microns using frozen cryostat. Free-‐Floa$ng Immunohistochemistry (IHC) • Double immunofluorescence with TH (LC marker
at 1:1000) and HT7 (hTau; 1:500), AT8 (hyperphosphorylated tau; 1:500), and MC1 (misfolded tau; 1:1000).
• Applied fluorescent secondaries gαM 546 (1:600) and gαRb 488 (1:600) to primary an0bodies.
Immunohistochemistry
Figure 1. Endogenous expression of P301S mutant hTau in the LC of Tau Tg mice. Coronal sec0ons of the LC of P301S-‐Tau mice were immunostained with TH (red) and P301S mutant hTau was immunostained with the human tau an0body HT7 (green) at all 0me points, including (A) 2 months and (B) 5 months of age.
P301S-‐Tau Tg mice express hTau in the LC from 2 mo. onwards
TH HT7 Merge
2 mo.
5 mo.
Eagan Ze^lemoyer1, Termpanit Chalermpalanupap2,3, Canaan Jerome3, David Weinshenker2,3 1College of Arts and Sciences, 2Neuroscience Graduate Program, 3Department of Human Gene0cs, Emory University, Atlanta, GA 30322 USA
3 mo.
TH Merge AT8
4 mo.
Conclusions
Future Direc$ons • Use more mice for more significant results. • Use different an0bodies for
hyperphosphorylated (i.e. AT100, AT180) and misfolded (i.e. PHF-‐1) tau.
• Inves0gate misfolded tau at later 0me points.
• Inves0gate when NFTs first appear and when the LC begins to degenerate.
References 1) Braak et al. (2011). Stages of the pathologic process in Alzheimer disease: age categories from 1 to 100 years. Na0onal Center for Biotechnology Informa0on. 2) Yoshiyama et al. (2007). Synapse loss and microglial ac0va0on precede tangles in a P301S tauopathy mouse model. Na0onal Center for Biotechnology Informa0on.
A
B
HT7 AT8 MC1 2 + -‐ -‐ 3 + -‐ -‐ 4 + + -‐ 5 + + -‐ M
onths o
f Age
An0bodies—TH (+)
Figure 2. Expression of hyperphosphorylated tau in the LC of Tau Tg mice. Coronal sec0ons of the LC of P301S-‐Tau mice were immunostained with TH (red) and hyperphosphorylated tau was immunostained with the abnormal tau an0body AT8 (green) at all 0me points, including (A) 3 months and (B) 4 months of age.
P301S-‐Tau Tg mice begin expressing abnormal tau in the LC at 4 mo.
2 mo.
5 mo.
TH MC1 Merge
P301S-‐Tau Tg mice do not express misfolded tau in the LC through 5 mo. of age
Figure 3. No expression of misfolded tau in the LC of Tau Tg mice. Coronal sec0ons of the LC of P301S-‐Tau mice were immunostained with TH (red) and misfolded tau was immunostained with the conforma0onal an0body MC1 (green) at all 0me points, including (A) 2 months and (B) 5 months of age.
A
A
B
B
• This manifesta0on of hyperphosphorylated tau in LC precedes its’ spread to the hippocampus recent studies have shown it is first seen at 6 mo. In this brain region.
• As expected, soluble hTau is expressed endogenously in the LC of all P301S-‐Tau Tg mice.
• Hyperphosphorylated tau is first detected in the LC of these mice at 4 mo. of age.
• Misfolded tau is not seen in the LC of these mice at all up through 5 mo. of age.
• P301S-‐Tau Tg mouse is a good model of hyperphosphorylated tau, for Braak et al. saw a similar progression of abnormal tau buildup in the LC of human young adults.
3) Takeuchi et al. (2011). “P301S Mutant Human Tau Transgenic Mice Manifest Early Symptoms of Human Tauopathies with Demen0a and Altered Sensorimotor Ga0ng.” PubMed. U.S. Na0onal Library of Medicine.