Dissertation Talk

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  • common symptom pattern begins with gradually worsening difficulty remembering new information. This is because disruption of brain cells usually begins in
    regions involved in forming new memories. As damage spreads, individuals also experience confusion, disorganized thinking, impaired judgment, trouble expressing themselves and disorientation.
  • a person has a problem with memory, language or another essential cognitive function serious enough to be noticeable to others and to show up on tests, but not severe enough to interfere with daily life. Some, but not all, people with MCI develop dementia over time, especially when their primary area of difficulty involves memory




  • Deshpande et al submitted manuscript

  • New slide for 4R

  • Dosage and time
  • Oligomer panel as a separate panel of animation
  • Oligomer panel as a separate panel of animation
























  • Bigger panels for this slide… chronic changes in mitochondrial function, but no cell death
    Previous studies indicate that Aß soluble species are toxic at low concentrations, reduce synaptic density, induce cognitive dysfunction, and inhibit long-term potentiation (Lambert et al., 1998; Hartley et al., 1999; Dahlgren et al., 2002; Walsh et al., 2002a; Wang et al., 2002).

  • We just saw what happens once AßO are at the synapses/near cell surface…

  • Change image color!!! Either one…
  • Change image color!!! Either one…
  • Change image color!!! Either one…
  • Change image color!!! Either one…
  • Change image color!!! Either one…
  • Change image color!!! Either one…








  • We just saw what happens once AßO are at the synapses/near cell surface…
  • We just saw what happens once AßO are at the synapses/near cell surface…
  • We just saw what happens once AßO are at the synapses/near cell surface…
  • We just saw what happens once AßO are at the synapses/near cell surface…
  • We just saw what happens once AßO are at the synapses/near cell surface…
  • We just saw what happens once AßO are at the synapses/near cell surface…
  • We just saw what happens once AßO are at the synapses/near cell surface…
  • We just saw what happens once AßO are at the synapses/near cell surface…

  • About 40 new cases of AD have developed and hopefully we are a step closer in understanding the mechanisms underlying AD pathology and to developing preventive therapeutics.




  • Dissertation Talk

    1. 1. Molecular Mechanisms Involved in Alzheimer’s Disease Pathology Atul Deshpande, Ph.D P.I.: Dr. Jorge Busciglio Dept. of Neurobiology and Behavior University of California, Irvine
    2. 2. Background: Prevalence  Alzheimer’s Disease is the leading cause of dementia among elderly people.  An estimate of 12% Americans over the age of 65 and almost half above the age of 85 have AD (~5 million).  Alzheimer’s Disease is the 3rd most expensive disease to treat, costing the society close to $100 billion annually.  AD is a progressive disorder beginning with memory deficits and leading to dementia.
    3. 3. Background: Etiology  The disease is characterized by two main lesions: Amyloid Plaques Neurofibrillary Tangles  Synaptic loss precedes neuronal loss  The neurodegeneration occurs in several brain areas including cortex, hippocampus and basal forebrain.
    4. 4. This Talk...
    5. 5. This Talk...  Aß - • Brief Background: Different conformations • Results • Effect of different conformations of Aß on HCN • Role in AD pathogenesis • Conclusions with possible model system for mechanism(s) of neurotoxicity
    6. 6. Beta Amyloid (Aß): main component of plaques  Two types of Aß deposits are found, a) the dense core neuritic plaques consisting of fibrillar insoluble Aß, and b) the diffuse plaques  A 40-42 amino acid peptide product of ß and γ secretase mediated cleavage of amyloid precursor protein (APP)  Fibrilization of Aß is preceded by multiple conformational changes but the pathways to oligomerization and fibrilization can be independent (Necula et. al., 2007)
    7. 7. Different Conformations of ß-amyloid Aβ42 Monomers Aß Derived Diffusible Ligands (ADDLs) Aβ42 Oligomer (AβO) AFM EM O L EM Aß*56 M O G I R E S (Lambert et. al., 1998) Demuro et. al. 2004 Annular Protofibrils Protofibrils EM Aβ42 Fibrils (AβF)
    8. 8. Different Conformations of ß-amyloid Aβ42 Monomers Aß Derived Diffusible Ligands (ADDLs) Aβ42 Oligomer (AβO) AFM EM O L EM Aß*56 M O G I R Soluble E S (Lambert et. al., 1998) forms Demuro et. al. 2004 of Aß Annular Protofibrils Protofibrils EM Aβ42 Fibrils (AβF)
    9. 9. Alzheimer’s Disease: The Different Faces of Amyloid-ß… Amyloid-ß Ravana Amyloid Precursor Protein
    10. 10. Alzheimer’s Disease: The Different Faces of Amyloid-ß… Amyloid-ß …and all of them are Evil!!!
    11. 11. Soluble Aß… M.W. AßO and ADDLs are found 100 AßO (~90kd) in the brains of AD patients 75 and have been found to… 50 a) Induce acute electrophysiological changes, 25 b) Inhibit long-term potentiation 20 ADDLs (~17-24kd) and impair memory function 15 c) Permeabilize cell membrane 10 and lead to influx of calcium, Monomer (~4.5kd) d) Affect neuronal viability
    12. 12. Objective of this study… Characterization of soluble oligomeric amyloid has raised the possibility that different Aß conformations may contribute to AD pathology via different mechanism(s) To characterize the toxicity of different Aß species: We tested side by side the effect of well characterized AßO, ADDLs and Aß fibrils in Human Cortical Neurons.
    13. 13. Experimental Model: Human Cortical Neurons (HCN)  Target Cells in Alzheimer’s Disease  Similar profiles of protein expression to the adult human brain (e.g. tau, etc.). HCN (0 DIV) HCN (20 DIV)
    14. 14. The most Sophisticated system ever designed…
    15. 15. The most Sophisticated system ever designed… By us!!! Control 0 4 8 12 hr hrs hrs hrs β-amyloid treated
    16. 16. The most Sophisticated system ever designed… By Zeiss!!!
    17. 17. Aß42f induce neuronal dystrophy, synaptic loss and modest cell death over an extended period of time Control AβF (20μM; 10 Days) Tau Aβ (Grace et.al;2002; Grace and Busciglio; 2003)
    18. 18. Soluble Aß colocalizes with synaptic markers in HCN 60 % colocolization 40 20 0 5 30 60 min AßO are rapidly targeted to synaptic terminals
    19. 19. AßO induces rapid and extensive neuronal death AßO (5µM) - 24 hours
    20. 20. AßO induces rapid and extensive neuronal death AßO (5µM) - 24 hours
    21. 21. AßO induce rapid mitochondrial alterations AßO at 5µM very rapidly activate an apoptotic cascade in HCN
    22. 22. Soluble AßOs induce translocation of cyt. c and AIF from mitochondria to the cytoplasmic compartment
    23. 23. AßO neurotoxicity under normal and low extracellular calcium After 12 hr with 5µM AßO… Regular Low Ca+2 medium ↓ mitochondrial oxido- No Change reductase activity ↑ activated caspases No Change 3/7 ↑ LDH in supernatant No Change After 24 hr with 5µM AßO… Regular Low Ca+2 medium ↓ mitochondrial oxido- ↓ mitochondrial oxido- reductase activity reductase activity ↑ activated caspases ↑ activated caspases 3/7 3/7 ↑ LDH in supernatant ↑ LDH in supernatant AßO neurotoxicity is accelerated by calcium influx
    24. 24. Incubation with AßO at lower concentrations Lower concentrations of AßO showed chronic mitochondrial alterations but no cell death
    25. 25. Summary… MMP: Mitochondrial Membrane Potential ATP: Energy currency of the Neurons MTS: Measure of efficiency of some of the key enzymes of the electron transport chain Cyt.C/AIF: Cytochrome C/Apoptosis Inducing Factor LDH: Lactate Dehydrogenase released/leaked into culture media
    26. 26. Mechanisms of AßO synaptic targeting a) Activity dependent AßO targeting? b) Role of metal ions? c) Endocytosis? d) Binding to specific receptor(s)?
    27. 27. Methods:  Two different culture systems were used: 1. Rat hippocampal organotypic cultures: allows the study of well characterized structures and synapses 2. HCN: allows the tracking of individual endocytic vesicles  Colocalization studies were carried out using three different antibodies (A11, NU2 and M69)  Z-sections were reconstructed using a rendering software to visualize the colocalization of AßO and synaptophysin
    28. 28. Neuronal activity modulates localization of AßO to synaptic terminals  Stimulation: 5min  20 mM KCl  100µM glutamate  Inhibition: 15 min 2µM TTX
    29. 29. Neuronal activity modulates localization of AßO to synaptic terminals  Stimulation: 5min  20 mM KCl  100µM glutamate  Inhibition: 15 min 2µM TTX min
    30. 30. Role of metal-ions: ✓ Metal ions, especially Cu+2 and Zn+2, are released at synaptic terminals during synaptic activity ✓ Aß binds to Cu+2 and Zn+2 with very high affinity (K1(Zn): 7; K1(Cu): 8.9) ✓ Thus, released metal-ions in the synaptic cleft could “attract” AßO to the synaptic terminals
    31. 31. Clioquinol: Effects of Oral Treatment of 15-Month-Old APP2576 Transgenic Mice with Clioquinol (from Cherny et. al., 2001) 5-chloro-7-iodo-quinolin-8-ol  Clioquinol, iodochlorhydroxyquin, is a metal-protein- attenuating compound (MPAC)  Clioquinol topical is an antifungal and antibacterial medication  Clioquinol is a chelator that allows the sequesstration of Zn/Cu-binding compounds, including Aß  A phase II trial reports that AD patients on the drug had a slower cognitive decline than patients on placebo (Ritchie CW, et. al., 2004)  Also effective to reduce htt accumulation in a Huntington’s mouse model
    32. 32. Effect of metal-ion chelation by clioquinol on synaptic localization of AßO Metal ions appear to be involved in synaptic localization of AßO
    33. 33. Analysis of AßO endocytosis at synaptic terminals AßO are not endocytosed at synaptic terminals
    34. 34. NMDAR activation appears to mediate synaptic localization of AßO • Recent studies have suggested role of specific receptors in AßO mediated neurotoxicity • NMDA receptor activity was inhibited using APV, memantine and ifenprodil. • AMPA receptor activity was inhibited using CNQX
    35. 35. NMDAR activation appears to mediate synaptic localization of AßO • Recent studies have suggested role of specific receptors in AßO mediated neurotoxicity • NMDA receptor activity was inhibited using APV, memantine and ifenprodil. • AMPA receptor activity was inhibited using CNQX AßO neurotoxicity appears to be mediated via NMDA receptor
    36. 36. Oligomeric specific antibody labeling colocalizes with synaptic markers in the AD Brain 8 NL and 10 AD cases were analyzed:  AßO labeling colocalized in 8 of 10 AD cases  Weak AßO labeling observed in 2 of 8 NL cases
    37. 37. Oligomeric specific antibody labeling colocalizes with synaptic markers in the AD Brain 8 NL and 10 AD cases were analyzed:  AßO labeling colocalized in 8 of 10 AD cases  Weak AßO labeling observed in 2 of 8 NL cases
    38. 38. Mechanisms of AßO synaptic targeting
    39. 39. Mechanisms of AßO synaptic targeting Activity dependent AßO targeting ✓
    40. 40. Mechanisms of AßO synaptic targeting Activity dependent AßO targeting ✓ Role of metal ions ✓
    41. 41. Mechanisms of AßO synaptic targeting Activity dependent AßO targeting ✓ Role of metal ions ✓ Endocytosis X
    42. 42. Mechanisms of AßO synaptic targeting Activity dependent AßO targeting ✓ Role of metal ions ✓ Endocytosis X Binding to specific receptor(s) ?
    43. 43. Working Model for AßO mediated molecular mechanism(s) in AD pathogenesis
    44. 44. Conclusion:  AßO affect synaptic plasticity and neuronal function  Multiple Aß species capable of deleterious effects at multiple levels coexist in the AD brain.  Therapeutic strategies to address Aß-mediated neurotoxicity will require a refined multimodal approach.
    45. 45. Alzheimer’s Disease Lesions: Amyloid Plaques Neurofibrillary Tangles
    46. 46. Alzheimer’s Disease Lesions: Amyloid Plaques Neurofibrillary Tangles Multimeric and oligomeric tau and Aß species appear to have a significant role in AD pathology Establishing how the load of Aß-soluble species changes during disease progression Exploring the role of specific receptors in AßO mediated neurotoxicity and relationship of AßO with post synaptic receptors in the AD brain
    47. 47. Acknowledgements P.I: Dr. Jorge Busciglio Supported by funding from NIH and Alzheimer’s Association, Hilblom Foundation and ADRC, UCI
    48. 48. Acknowledgements P.I: Dr. Jorge Busciglio Lab Members Dr. Gustavo Pigino Ale Pelsman Pablo Helguera Khin Win Michael Hanna Rosa Resende Jackie Selgie Octavio Garcia Ardy Rahman Supported by funding from NIH and Alzheimer’s Association, Hilblom Foundation and ADRC, UCI
    49. 49. Acknowledgements P.I: Dr. Jorge Busciglio Lab Members Collaborators: Dr. Gustavo Pigino Dr. Charlie Glabe Ale Pelsman Glabe Lab Special Thanks Pablo Helguera Dr. Frank LaFerla Dr. Raju Metherate Khin Win LaFerla Lab Dr. Hideki Kawai Michael Hanna Dr. Katumi Sumikawa Dr. John Marshal Rosa Resende Dr. Ian Parker Dr. Amit Deshpande Jackie Selgie Dr. Angelo Demuro Octavio Garcia Dr. Marcelo Wood Ardy Rahman Dr. Gustavo Pigino, UIC Dr. Scott Brady, UIC Supported by funding from NIH and Alzheimer’s Association, Hilblom Foundation and ADRC, UCI

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