THE AUSTRALIAN NATIONAL UNIVERSITY

1. THE AUSTRALIAN NATIONAL UNIVERSITY Alzheimer’s DiseaseChristian StrickerANUMS/JCSMR - ANUChristian.Stricker@anu.edu.auhttp://stricker.jcsmr.anu.edu.au/Alzheimer.pptx

3. Aims The students should • know why AD is a neurodegenerative disease; • be aware of what makes up plaques & tangles; • obtain a cell-biological understanding of recent research findings and topics into AD; • appreciate why Aβ and presenilins have multiple actions, both physio- and pathologically; and • understand why AD is largely a synaptic disease.

4. Contents • Historical note: plaques & tangles • Production of plaques as Aβ oligomers • Tangles and axonal transport • Theory of Aβ cascade and proteolysis • Role of APP • Role of presenilins • Synaptic disease • Reduction in synaptic efficacy • Spine loss and NMDA receptors

5. History • 25. 11. 1901: Alfred Alzheimer meets Auguste Deter, 51 yo, in Frankfurt. • 8. 4. 1906: Auguste D. dies. • 1906f: Alzheimer publishes first description of this illness with pathological findings (see later). • 1910: Term “Alzheimer’s disease” coined by Kräpelin (German psychiatrist). • “Forgetting”.

6. Plaques and Tangles Sisodia & St.George-Hyslop (2004), Nat. Rev. Neurosci. 3: 281-290 • Plaque: β-amyloid (Aβ) in centre and dystrophic nerve terminals filled with hyperphosphorylated tau around it. • Tangle: hyperphosphorylated microtubule-associated protein tau assembled into paired helical filaments.

7. Biochemistry of Plaques • Not only in nerve tissue, but also around blood vessels. • Consist of extracellular protein. • β-amyloid (~40 AA; Aβ) in “precipitated” form. • Assembly of Aβ oligomers. • Proteolyticproduct from amy-loid precursor protein (APP). • High content of heavy metals (Al2+, Zn2+, Cu2+; role?). • Related to amyloid in pancreas in diabetes-2 (adult onset). Van Dorpe et al. (2004), Am. J. Pathol. 157: 1283-1298

8. Emergence of Plaques • How long does it take to form a plaque? • Tracked in triple-TG mice using in vivo 2-photon microscopy. • Can emerge within a few days; followed by • microglia activation & recruitment (1 – 2 d); • progressive neuritic changes, leading to increasingly dysmor-phicneurites (d–w). Meyer-Luehmann et al. (2008), Nature 451: 720-724

9. Biochemistry of Tangles http://www.hia.nih.gov • Tau protein stabilizes neurofilaments via cross-linking subunits. • If hyperphosphorylated, tau assembles into tangles (intracellular). • As a consequence, neurofilaments destabilise and break up. • Axonal transport of mitochondria to/from synapse breaks: mitochondrial energy and Ca2+ homeostasis crisis at synapse.

10. Axonal Transport and Mitochondria Miller & Sheetz (2006), J. Cell Biol. 173: 373-381

11. Genetics versus Environment Cummings (2004), NEJM 352: 862-866 • Genetic link only for ~1% (autosomal dominant – familial AD, i.e. FAD). • Rest (~99%) sporadic (SAD); most likely a very heterogeneous group. • FAD has early onset (< 65 y) sharing 3 genes: APP, PS1, PS2. Mutation of one of these genes alone is sufficient to cause FAD. • SAD risk factors similar to atherosclerotic disease (BP, lipids, etc). • Disease with important genetic components.

12. Theories of Disease Generation Schon & Area-Gomez (2013), Mol Cell Neurosci55: 26-36 • No theory is proven… chicken & egg problem. • MAM – Mitochondria-associated membrane – lipid raft containing important signalling proteins (PSEN, BACE, etc.). • Production versus clearance problem (?) Extended from de la Torre (2004), Lancet Neurol3: 184-190

13. β-Amyloid Generation • Extracellular/intravesicularspace: 2 paths - one on right produces toxic Aβ. • also in secretory vesicles • If APP proteolysis done via α-secretase, NO Aβ is produced. • Amyloidogenic proteolysis: β-secretase cleaves head. • γ-secretase cleaves within membrane. Leads to Aβ production (40, 42 AA). • Particularly, Aβ42 is neurotoxic: • Aggregation • Excitotoxicity • Oxidation • Inflammation • Tau hyperphosphorylation Cummings (2004), NEJM 351: 56-67

14. What Is APP’s Physiological Role? • Ferro-oxidase function like coerulo-plasmin (Fe2+ oxidation to Fe3+) • Loads Fe3+ into transferrin (Fe homeostasis disease ?): export activity. • Free radical generation → oxid. stress. • Inhibited by Zn2+. • Amine oxidase activity: NA, A, DA↑. • All 3 proteolytic parts have role (?). • Head (N-APP): Development. • Axonal pruning via DR6 that activa-tescaspase 6: Apoptotic pathway. • Aβ (pathogenic part) • As monomer, secreted (synaptic activity): maintains synapses (trophic factor?). • As oligomers, forms amyloid sheets in vesicles (storage?). • Tail (AICD) • Calcium homeostasis. • ATP availability (mitochondria). • Transcriptional modulation. Nicholson (2009), Nature 457: 970-971

15. Properties of Aβ • Produced in ER; stored in vesicles. • Monomers may be neurotrophic. • Aβsecretion controlled by orexin; under circadian control: sleep ↓; awake ↑. • Production modulated by GPR3 (orphan receptor). • Binds to ACh receptor (pathology). • Oligomers are toxic, esp. Aβ42 • Aβ40 by cleavage in ER; Aβ42 in trans-Golgi network. • As peptides, ability to form cross-β-sheets (“amyloid”), which are poorly soluble. http://commons.wikimedia.org

16. “Players” in Amyloid Hypothesis • γ-secretase (multi-protein) complex consisting of • nicastrin, • presenilins(PS1, PS2), and • Aph-1. • γ-secretase also activatesnotchsignalling pathway (development; price to pay??). • Aβ self-assembles into oligomers, which become poorly soluble. • Over time, Aβ accumula-tion depends on produc-tion and clearance. Roberson & Mucke (2006), Science 314: 781-784

17. Presenilins and Ca2+ Homeostasis • Catalyse (as part of γ-se-cretase) formation of Aβ. • Central role in Ca2+ homeostasis (~ 100 nM): • modulate (in SER membr.) • SERCA pump • IP3 receptor • Ryanodine receptor • interact with Ca2+ binding proteins: calpain, etc. • form Ca2+ channels (in expression system): SER Ca2+ leak channel. • are essential for regulating transmitter release (presynaptic Ca2+ stores). • Ca2+ store release activates β-secretase. Green & LaFerla (2009), Neuron 59: 190-194

18. Functional Studies What is Affected? What are the Consequences?

19. AD - A Synaptic Disease • Several transgenic mouse models (overexpression). • Human APP mutation transgenic mouse (overexpression). • Early onset in hippocampus (2-10 mo.). • Number of synapses↓. • Number of cells↓. Hsia et al. (1999), PNAS 96: 3228-3233

20. Changes in Synaptic Efficacy • AMPA-type fEPSPs reduced. • NMDA-dependent LTP does not seem to be affected (other reports differ…). • Larger NMDA receptor currents in transgenic animals. • Potential source of excitotoxicity via Ca2+ “overload”. Hsia et al. (1999), PNAS 96: 3228-3233

21. “Neurotoxicity” • Organotypic slice cultures of CA1 hippocampus. • Spines are sites of excitatory synapses. • When exposed to control and Aβ42 monomers, spine density is normal. • When exposed to Aβ42oligomers, spine density drops. Shankar et al. (2007), J. Neurosci. 27: 2866-2875

22. Aβ Secretion Is Activity Dependent • Acutely transfected organotypic hippocam-pal slice cultures (virus). • Neuronal activity secretes Aβ: • TTX blocks APs. • High Mg2+“blocks” transmission. • Picrotoxin (PTX) blocks GABAA receptors (disinhibition). • Flunitrazepam is a GABAA receptor modulator. Kamenetz et al. (2007), Neuron 37: 925-937

23. Link to Prion-Protein (PrPc) • Neurodegenerative diseases show similar histology. • Aβ42 released at synapses. • Genome-wide screen for Aβoligomers reveals PrPc. • Postsynaptic PrPc lipid rafts. • Functional studies • LTP↓ in wild-type mice. • LTP normal in PrPc KO mice. • α-secretase cleaves PrPc. • Tau, too, complexes with PrPc (postsynaptic toxicity of Aβ – as a Trojan invader?). • PrPc may be good drug target • KO mice viable and LTP normal. Cisse & Mucke (2009), Nature 457: 1090-1091

24. How is all this in vivo? Still a lot of work to do…

25. Take-Home Messages • Few cases with genetic background. • More theories than understanding (MAM “disease”). • Plaques are made out of Aβ-oligomers. • Tangles contain of hyperphosphorylated tau. • AD is largely a synaptic disease: • Fewer cells and synapses. • Synapses with smaller efficacy. • Larger contribution of NMDA receptor currents. • NMDA receptor-dependent spine loss. • Activity dependent Aβ secretion.

26. References • Textbooks • Kandel (4th ed.): 1149 - 1161 • Reviews • Cummings JL (2004) Alzheimer's Disease. N Engl J Med 351: 56-67. • Roberson ED, Mucke L (2006) 100 Years and Counting: Prospects for Defeating Alzheimer's Disease. Science 314: 781-784. • Goedert M, Spillantini MG (2006) A Century of Alzheimer's Disease. Science 314: 777-781. • Selkoe DJ (2002) Alzheimer's Disease Is a Synaptic Failure. Science 298: 789-791. • Blennow K, de Leon MJ, Zetterberg H (2006) Alzheimer's Disease. Lancet 368: 387-403. • de la Torre JC (2004) Is Alzheimer's Disease a Neurodegenerative or a Vascular Disorder? Data, Dogma, and Dialectics. Lancet Neurol 3: 184-190.

27. That’s it folks…