Neuronal Cell Death

Background:

Neurons are damaged or lost as a consequence of injury and disease. This can significantly alter our capacity to utilize the five senses, our ability to ambulate, and our behavior and cognition. Neuronal damage has been associated with diverse forms of injury and disease including stroke, trauma, seizures, AIDS and neurodegenerative diseases. It is not presently clear how these various forms of injury and disease compromise the function and viability of neurons. If an insult to the nervous system is very intense, then neurons may die by a passive, energy-independent process referred to as necrosis (see figure 1). If the insult is of less intensity, then neurons die by an energy dependent process requiring the activation of discrete genetically programmed pathways. This active form of cell death is referred to as apoptosis. Whereas neurons may die by necrosis in a matter of hours, the process of apoptosis may occur over the course of days to weeks. As neurons undergo necrosis they swell and eventually burst dispersing their intracellular constituents into the extracellular space consequently inciting an inflammatory response. Inflammation fosters more damage and cell death in the nervous system. In contrast, apoptosis involves the ordered breakdown of a cell with its ultimate shrinkage and elimination by professional scavengers, minimizing the inflammatory process. However, any insult that produces neuronal apoptosis ultimately eliminates these cells and compromises the function of the brain or spinal cord. Our laboratory has been characterizing the biochemical pathways involved in neuronal apoptosis caused by injury and disease.

Our ultimate goal is to preserve the structural and functional integrity of the brain and spinal cord by blocking the biochemical pathways that culminate in neuronal cell death following injury and disease.

Research Question:

Are there master switches regulating the neuronal response to injury and disease?

Neurons respond to stress in different ways depending on the nature of the injury or disease. Although different types of injury and disease may stimulate diverse pathways in neurons, there are some uniform biochemical responses to cellular stress. For example, one common response to neuronal damage is the production of reactive oxygen species. These are highly reactive molecules that bind and damage proteins, lipids and DNA. All cells possess a surveillance mechanism for identifying and then repairing DNA damage. One such molecule involved in this process is the p53 tumor suppressor, which is a nuclear protein that functions as a key regulator of DNA repair, cell cycle progression and apoptosis. When growing cells acquire damage to their genetic material, p53 essentially applies a brake so they no longer divide and can repair DNA damage prior to dividing into new cells. As a result, p53 ensures that cells do not pass on genetic mutations that might eventually cause cancer. In fact, loss or inactivation of the p53 tumor suppressor gene occurs in almost half of all human tumors and is considered a fundamental, predisposing event in the pathogenesis of many cancers.

What is seemingly a contradiction in our understanding of cell death in post-mitotic neurons is the demonstration that neuronal damage results in the elaboration of events that are only associated with proliferating cells. Surprisingly, p53 expression is elevated in damaged neurons in acute models of injury such as ischemia and epilepsy and in brain tissue samples derived from animal models and patients with chronic neurodegenerative diseases. The absence of p53 in genetically engineered mice or the application of p53 inhibitors protects neurons from a wide variety of acute toxic insults. Signal transduction pathways associated with p53-induced neuronal cell death are being characterized, and their modulation has been shown to protect neurons from varying forms of cellular stress. This suggests that interrupting the function of p53 may prove effective in maintaining neuronal viability and restoring function following neural injury and disease.

For Research Highlight details, click on the red links below:

Research Highlights:

I. Evidence for p53-Mediated Modulation of Neuronal Viability

II. Bax Involvement in p53-mediated Neuronal Cell Death

III. p38-MAP Kinase Mediates Bax Translocation During Neuronal Apoptosis

IV. Peg3 Is A Mediator Between p53 and Bax In DNA Damage-Induced Neuronal Death

V. Mass Spectrometric Proteome Analysis of p53-dependent Neuronal Death

VI. Using new technologies to study the p53 gene's role in neurodegenerative and neurodevelopmental disorders (pdf file)

Research Methods:

Our laboratory utilizes a wide variety of cell and molecular biology approaches to study neuronal cell death:

• Primary Neuronal and Glial Cultures
  
  
• Animal models;
  - Kainate-induced seizures
  - MPTP- a model of Parkinson’s disease
  - Various knockout mice are used to evaluate the role of apoptotic mediators
  
  
• Adenovirus production, purification and transduction
  
  
• Traditional biochemical techniques;
  - Polyacrylamide gel electrophoresis
  - Western blot
  - PCR Protein, mRNA and DNA purification
  - Caspase cleavage assays
  
  
• Immunostaining and Imaging;
  - Fluorescent and bright field imaging on fixed and live cells and tissues
  
  
• cDNA microarray
  
  
• Proteomics;
  - ICAT labeling
  - HPLC
  - Mass Spectrometry
  - Bioinformatics
  
  

Present Lab Members:

- Chizuru Kinoshita
- Yoshito Kinoshita, Ph.D.
- Takuma Uo, Ph.D.
- Lidong Liu, Ph.D.
- Camelia Danilov, Ph.D.
- Cody Wyles
- Bonita Lee

Previous Lab Members:

- Jenny Dworzak
- Matthew Batten
- Laura Andrews
- Gwenn Garden, M.D., Ph.D. (Collaborator)
- Elise Bergelson, M.D.
- Joseph Ho
- Saadi Ghatan, M.D.
- Suzanne Giordano, Ph.D.
- Mary Lee-Hanson, Ph.D.
- Joshua McBee, Ph.D.
- Abel Jarell, M.D.
- Charlie Kuntz, M.D.
- Stephen Larner, M.S.
- Mark Johnson, M.D., Ph.D.
- Susumu Nagasaka, M.D.
- Jim Schuster, M.D., Ph.D.
- Mark Tang
- Hong Xiang, M.D.
- Alan Yahanda, M.D.
- Shoko Yamada, M.D.
- Fumio Yamaguchi, M.D.

Bibliography:

SELECTED PUBLISHED AND ACCEPTED ARTICLES IN REFEREED JOURNALS:

1. Morrison, R.S.,Wenzel, H.J., Kinoshita, Y., Robbins, C.A.,Donehower, L.A., Schwartzkroin, P.A. Loss of the p53 Tumor Suppressor Gene Protects Neurons From Kainate Induced Cell Death. J. Neurosci.16:1337-1345, 1996.
2. Xiang, H., Hochman, D.W., Saya, H., Fujiwara, T., Schwartzkroin, P.A. and Morrison, R.S.: Evidence for p53-Mediated Modulation of Neuronal Viability. J. Neurosci., 16:6753-6765, 1996.
3. Xiang, H., Kinoshita, Y., Knudson, C. M., Korsmeyer, S.J., Schwartzkroin, PA. and Morrison, R.S. Bax involvement in p53-mediated neuronal cell death. J. Neurosci., 18:1363-1373, 1998.
4. Johnson M.D., Kinoshita Y., Xiang H., Ghatan S. and Morrison R.S. Contribution of p53-dependent caspase activation to neuronal cell death declines with neuronal maturation. J. Neurosci., 19:2996-3006, 1999.
5. Ghatan S., Larner S., Kinoshita Y., Hetman M., Patel L., Xia Z., Youle R.J. and Morrison R.S. p38 Mitogen-activated Protein Kinase Mediates Bax translocation and Caspase Induction in Nitric Oxide-induced Apoptosis in Neurons. J. Cell Biol., 150:335-348, 2000.
6. Morrison R.S. and Kinoshita Y. p73:Guilt by Association? Science, 289:257-258, 2000.
7. Kuntz C., Kinoshita Y., Beal F., Donehower LA, and Morrison R.S. The absence of p53 does not protect SOD1 mutant mice from onset of clincial symptoms or lethality. Exp. Neurol., 165:184-190, 2000.
8. Morrison R.S. and Kinoshita Y. The Role of p53 in Neuronal Cell Death. Cell Death and Diff., 7:868-879, 2000.
9. Kinoshita Y., Jarell A.D., Flaman J.M., Foltz G., Schuster J., Sopher B.L., Kanning K., Irvin D.K., Kornblum H.I., Nelson P.S., Hieter P. and Morrison R.S. Pescadillo, a Novel Cell Cycle Regulatory Protein Abnormally Expressed in Malignant Cells. J. Biol. Chem., 276:6656-6665, 2001.
10. Garden GA, Libby RT, Fu Y-H, Kinoshita Y, Einum DD, Huang J, Possin DE, Morrison RS, Ptacek LJ, Sopher BL, and La Spada AR. Neurological dysfunction in a mouse model of SCA7 is associated with proteolytic cleavage of polyglutamine-expanded ataxin-7. J. Neurosci, 22:4897-4905, 2002.
11. Johnson M.D., Wu X., Aithmitti N. and Morrison R.S. Peg3 Is A Mediator Between p53 and Bax In DNA Damage-Induced Neuronal Death. In Press, J. Biol. Chem., 2002.
12. Li-Rong Y., Johnson M.D., Conrads T.P., Morrison R.S. and Veenstra T.D. Isotope-coded Affinity Tag Analysis of Native and Camptothecin-treated Cortical Neurons. Electrophoresis, 23:1591-1598, 2002.
13. Morrison R.S., Kinoshita Y., Johnson M.D., Guo W. and Garden G.A. p53-dependent cell death signaling in neurons. Neurochem Res, 28:15-27, 2003.
14. Yu L-R., Issaq H.J., Conrads T.P., Uo T., Blonder J., Janini G.M., Morrison R.S. and Veenstra T.D. Evaluation of Liquid Chromatography-Mass Spectrometry for Routine Proteome Analyses. Journal of Liquid Chromatography & Related Technologies, 26(20):3325-3336, 2003.
15. Yu L-R., Conrads T.P., Uo T., Issaq H.J., Morrison R.S. and Veenstra T.D. Evaluation of the acid-cleavable isotope-coded affinity tag reagents: application to camptothecin-treated cortical neurons. Journal of Proteome Research, 3:469-477 2004.
16. Johnson M.D., Li-Rong Y., Conrads T.P., Kinoshita Y., Uo T., Lee S.W., Smith D., Veenstra T., and Morrison R.S. Proteome Analysis of DNA Damage-Induced Neuronal Death Using High Throughput Mass Spectrometry. J. Biol. Chem., 279:26685-26697, 2004.
17. Garden G.A., Guo W., Tun C., Jayadev S., Balcaitis S., Moeller T., and Morrison R.S.. HIV Associated Neurodegeneration Requires p53 in Neurons and Microglia. FASEB J., 18(10):1141-1143, 2004. For Full Text: http://www.fasebj.org/cgi/doi/10.1096/fj.04-1676fje
18. Yu LR, Conrads T.P., Uo T., Kinoshita Y., Morrison R.S., Lucas D.A., Chan K., Blonder J., Issaq H.J., and Veenstra T.D. Global Analysis of the Cortical Neuron Proteome. Mol. and Cell. Proteomics, 3:896-907, 2004.
19. Garden G.A. and Morrison R.S. The Multiple Roles of p53 in the Pathogenesis of HIV Associated Dementia. BBRC, 331:799-809, 2005.
20. Uo. T., Kinoshita Y., and Morrison R.S. Neurons exclusively express an alternatively spliced BH3 domain only Bak isoform, N-Bak, that promotes neuronal apoptosis. J. Biol. Chem., 280:9065-9073, 2005.
21. Veenstra T.D., Conrads T.P., Hood B.L., Avellino A.M., Ellenbogen R.G., and Morrison R.S. Biomarkers: Mining the Biofluid Proteome. Molecular and Cellular Proteomics. 4:409-418, 2005.
22. Johnson M.D., Yu L-R, Conrads T.P., Kinoshita Y., Uo T., McBee J.K., Veenstra T.D., and Morrison R.S. The Proteomics of Neurodegeneration. American Journal of PharmacoGenomics., 5:259-70, 2005.
23. La Spada A.R. and Morrison R.S. The Power of the Dark Side: Huntington's Disease Protein and p53 Form a Deadly Alliance. Neuron, 47: 1-3, 2005.
24. Sikorski E.M., Uo T., Morrison R.S., and Agarwal A. Pescadillo interacts with the cadmium response element of the Human heme oxygenase-1 promoter in renal epithelial cells. J. Biol. Chem., 281:24423-30, 2006.
25. Kinoshita Y. Uo T., Jayadev S., Garden G.A., Conrads T.P., Veenstra T.D. and Morrison, R.S. Potential Applications and Limitations of Proteomics in the Study of Neurological Disease. Archives of Neurology, 63:1692-1696, 2006.
26. Tun C., Guo W., Nguyen H., Yun B., Libby R.T., Morrison R.S., Garden G.A. Activation of the extrinsic caspase pathway in cultured cortical neurons requires p53-mediated down-regulation of the X-linked inhibitor of apoptosis protein to induce apoptosis. J. Neurochem., 102:1206-1219, 2007.
27. McBee J.K., Yu L-R, Kinoshita Y, Uo T., Beyer R.P., Veenstra T.D., and Morrison R.S. Proteomic Analysis of Protein Expression Changes in a Model of Gliomagenesis. Proteomics Clin. Appl., 1:1485–1498, 2007.
28. Uo T., Kinoshita Y. and Morrison R.S. Apoptotic actions of p53 require transcriptional activation of PUMA and do not involve a direct mitochondrial/cytoplasmic site of action in postnatal cortical neurons. J. Neurosci, 27:12198-12210, 2007.
29. Jayadev S., Yun B., Nguyen H., Yokoo H., Morrison R.S. and Gwenn A. Garden. The Glial Response to CNS HIV Infection Includes p53 Activation and Increased Expression of p53 Target Genes. J. Neuroimmune Pharmacology, 2:359-30, 2007.