AGING, OXIDATIVE STRESS AND CELL DEATH

Release Date:  March 23, 2000

PA NUMBER:  PA-00-081

National Institute on Aging

THIS PA USES THE "MODULAR GRANT" AND "JUST-IN-TIME" CONCEPTS.  IT INCLUDES 
DETAILED MODIFICATIONS TO THE STANDARD APPLICATION INSTRUCTIONS THAT MUST BE 
USED WHEN PREPARING APPLICATIONS IN RESPONSE TO THIS PROGRAM ANNOUNCEMENT.

PURPOSE

The purpose of this Program Announcement (PA) is to encourage the submission 
of applications to support research on the relationship between oxidative 
stress and apoptosis, and how these biological processes are involved in 
aging and/or change with age.  This program announcement supersedes two PAs 
issued earlier by the NIA:  Molecular mechanisms of cell death during aging, 
and PA-93-017, Oxidative damage, antioxidant defense, and aging.

Although the National Institute of General Medical Sciences is not 
participating in this program announcement, that Institute continues to be 
interested in receiving applications on basic mechanisms of programmed cell 
death and oxidative stress when these applications do not focus on aging 
mechanisms.

HEALTHY PEOPLE 2010

The Public Health Service (PHS) is committed to achieving the health 
promotion and disease prevention objectives of "Healthy People 2010," a PHS 
led national activity for setting priority areas. This Program Announcement, 
Aging, oxidative stress, and cell death, is related to one or more of the 
priority areas. Potential applicants may obtain a copy of "Healthy People 
2010" at http://www.health.gov/healthypeople/

ELIGIBILITY REQUIREMENTS

Applications may be submitted by domestic and foreign for-profit and non-
profit organizations, public and private, such as universities, colleges, 
hospitals, laboratories, units of state and local governments, and eligible 
agencies of the Federal government. Racial/ethnic minority individuals, 
women, and persons with disabilities are encouraged to apply as principal 
investigators.

MECHANISM OF SUPPORT

The mechanism of support will be the National Institutes of Health (NIH) 
individual research project grant (R01).  Responsibility for the planning, 
direction, and execution of the proposed project will be solely that of the 
applicant.  The total project period for an application submitted in response 
to this PA may be up to 5 years.  For R01 applications requesting up to 
$250,000 direct costs per year, specific application instructions have been 
modified to reflect "MODULAR GRANT" and "JUST-IN-TIME" streamlining efforts 
being examined by NIH.  Complete and detailed instructions and information on 
Modular Grant applications can be found at: 
http://grants.nih.gov/grants/funding/modular/modular.htm. Applications that 
request more than $250,000 in any year must use the standard PHS 398 (rev. 
4/98) application instructions. 

FUNDS AVAILABLE

Applications submitted in response to this Program Announcement, and assigned 
to the NIA, will compete with all other applications assigned to the NIA.  No 
funds have been set aside for funding applications submitted in response to 
this PA.  The award of grants pursuant to this PA is contingent upon the 
availability of funds for this purpose and the receipt of meritorious 
applications.

RESEARCH OBJECTIVES

Cell death in multicellular organisms takes place in a variety of different 
circumstances.  For example, the death of certain cells may occur as part of 
a developmental program e.g. during nervous system development; in connection 
with organ involution or regression; when potentially harmful cells such as 
neoplastic or virus-infected cells are targeted for destruction by 
surveillance systems; in response to the absence of critical growth factors; 
or as a consequence of toxic or harmful environmental conditions.  When cells 
are damaged in mitotically active tissues, they can usually be replaced by 
division of neighboring healthy cells.  However, when post-mitotic cells are 
damaged and die, cell replacement may not be possible and the function of the 
tissue is eventually compromised, as occurs in the neurodegenerative 
disorders such as Alzheimer’s and Parkinson’s diseases.

Oxidative damage due to the endogenous production of reactive oxygen species 
by mitochondria is a common and ubiquitous form of oxidative stress in most 
mammalian cells.  However, these cells have robust systems for both detecting 
and repairing this damage, particularly in the case of DNA.  When confronted 
by damaged DNA, cells either set about repairing themselves or commit suicide 
through apoptosis.  The checkpoint proteins p53 and ATM (ataxia 
telangiectasia mutated) are designed to block DNA replication of damaged DNA 
until repair of the DNA has been accomplished (Weinert, 1998), and ATM 
deficiency results in the lack of p53 induction by ionizing radiation, a DNA 
damaging agent (Barlow et al., 1997).  When these checkpoint proteins are 
missing or defective, the result may be inappropriate cell growth, as in 
cancer (Levine, 1997; Venkitaraman, 1999), or inappropriate cell loss, as in 
ataxia telangiectasia.  Alternatively, the damage may be so extensive as to 
preclude adequate repair, leading to a variety of degenerative diseases.

It is now clear that cell death, especially apoptotic cell death, plays a 
role in a large variety of age-related pathological changes (Warner et al., 
1997).  Thus, understanding how a mammalian cell recognizes and repairs 
damage, and thereby maintains homeostasis, or conversely fails to repair the 
damage, is critical to understanding the biological basis of aging in a 
variety of cells and tissues.  In fact, fibroblasts taken from individuals 
with Werner syndrome, a premature aging syndrome, have an attenuated p53-
mediated apoptotic response (Spillare et al., 1999).  The importance of this 
question is further highlighted by the recent paper showing that a mutation 
in the p66shc protein not only blocks induction of oxidative repair functions 
and increases the resistance of the mice to paraquat, but also extends the 
life span of the mice by 30% (Migliaccio et al., 1999).

Also of particular interest is the role of mitochondria in these processes.  
Mitochondria are not only a major source of endogenous production of reactive 
oxygen species, but the release of cytochrome C from mitochondria also 
induces the entire caspase cascade involved in apoptosis (Slee et al., 1999).  
Furthermore, critical mitochondrial proteins involved in production of 
cytosolic ATP appear to be targets of increasing oxidative damage during 
aging (Yan et al., 1997), but it is not clear if and how this self-inflicted 
damage actually leads to mitochondrial leakage of cytochrome C and induction 
of apoptosis.  In neurons, following neurotrophic factor removal, a key event 
is the translocation of BAX from the cytosol to the mitochondria with 
subsequent release of cytochrome C and activation of caspases (Putcha et al., 
1999).  Mitochondrial dysfunction due to oxidative damage and leading to cell 
death may thus be a unifying basic mechanism involved in neurodegeneration 
(Beal, 1998).

The major objectives of this program announcement include, but are not 
limited to applications designed to address one or more of the following 
research questions:

o  Are there age-related changes in apoptotic potential?
o  What is the role of apoptosis in adverse age-related changes?
o  How does damage to DNA, proteins and lipids induce the apoptotic process?
o  Under what conditions, if any, is cell death preferable to survival of a 
damaged cell?
o  How does mitochondrial dysfunction contribute to both oxidative stress and 
apoptosis?
o  Identification of single nucleotide polymorphisms in genes such as those 
for ATM, p53, caspases, bcl-2 family members, caspase inhibitors, and DNA 
repair enzymes, and/or their influence on any of the above questions.
o  Use of transgenic and/or knockout mice to evaluate the role of any of 
these gene products in oxidative stress, mitochondrial dysfunction, 
macromolecular repair processes and apoptosis, with special emphasis on the 
role of these processes in aging.

REFERENCES

Barlow, C. et al.  1997.  ATM selectively regulates distinct p53-dependent 
cell-cycle checkpoint and apoptotic pathways.  Nature Genetics 17: 453-456.

Beal, M.F.  1998.  Mitochondrial dysfunction in neurodegenerative diseases.  
Biochem. Biophys. Acta 1366: 211-223.

Levine, A.J.  1997.  p53, the cellular gatekeeper for growth and division.  
Cell 88: 323-331.

Migliaccio, E., et al.  1999.  The p66shc adaptor protein controls oxidative 
stress response and life span in mammals.  Nature 402: 309-313.

Putcha G.V., et al.  1999.  BAX translocation is a critical event in neuronal 
apoptosis:  regulation by neuroprotectants, BCL-2, and caspases.  J. 
Neurosci. 19: 7476-7485.

Slee, E.A., et al.  1999.  Ordering the cytochrome C-initiated caspase 
cascade:  Hierarchial activation of caspases-2, -3, -6, -7, -8 and –10 in a 
caspase-9-dependent manner.  J. Cell Biol. 144: 281-292.

Spillare, E.A. et al.  1999, p53-mediated apoptosis is attenuated in Werner 
syndrome cells.  Genes Dev. 13: 1355-1360.

Venkitaraman, A.R.  1999.  Breast cancer genes and DNA repair.  Science 286: 
1100-1102.

Warner, H.R., et al.   1997.  What does cell death have to do with aging?  J. 
Am. Ger. Soc. 45: 1140-1146.

Weinert, T.  1998.  DNA damage and checkpoint pathways:  Molecular anatomy 
and interactions with repair.  Cell 94: 555-558.

Yan, L.-J., et al., 1997.  Oxidative damage during aging targets 
mitochondrial aconitase.  Proc. Natl. Acad. Sci. USA, 94: 11168-11172.

INCLUSION OF WOMEN AND MINORITIES IN RESEARCH INVOLVING HUMAN SUBJECTS

It is the policy of the NIH that women and members of minority groups and 
their sub-populations must be included in all NIH supported biomedical and 
behavioral research projects involving human subjects, unless a clear and 
compelling rationale and justification is provided that inclusion is 
inappropriate with respect to the health of the subjects or the purpose of 
the research.  This policy results from the NIH Revitalization Act of 1993 
(Section 492B of Public Law 103-43).

All investigators proposing research involving human subjects should read the 
"NIH Guidelines For Inclusion of Women and Minorities as Subjects in Clinical 
Research," which have been published in the Federal Register of March 28, 
1994 (FR 59 14508-14513) and in the NIH Guide for Grants and Contracts, 
Volume23, Number 11, March 18, 1994, 
http://grants.nih.gov/grants/guide/notice-files/not94-100.html. 

Investigators also may obtain copies of these policies from the program staff 
listed under INQUIRIES. Program staff may also provide additional relevant 
information concerning the policy.

APPLICATION PROCEDURES

Applications are to be submitted on grant application form PHS 398 (rev. 
4/98). and will be accepted at the standard application deadlines as 
indicated in the application kit. Application kits are available at most 
institutional offices of sponsored research and may be obtained from the 
Division of Extramural Outreach and Information Resources, National 
Institutes of Health, 6701 Rockledge Drive, MSC 7910, Bethesda, MD 20892-
7910, Phone (301) 435-0714, Email:  GRANTSINFO@NIH.GOV. Applications are also 
available on the internet at 
http://grants.nih.gov/grants/funding/phs398/phs398.html.

Applicants planning to submit an investigator-initiated new (type 1), 
competing continuation (type 2), competing supplement, or any amended/revised 
version of the preceding grant application types requesting $500,000 or more 
in direct costs for any year are advised that they must contact the Institute 
or Center (IC) program staff before submitting the application, i.e., as 
plans for the study are being developed. Furthermore, applicants must obtain 
agreement from the IC staff that the IC will accept the application for 
consideration for award. Finally, applicants must identify, in a cover letter 
sent with the application, the staff member and Institute or Center who 
agreed to accept assignment of the application.

This policy requires applicants to obtain agreement for acceptance of any 
such application and/or any such subsequent amendment. Refer to the NIH Guide 
for Grants and Contracts, March 20, 1998 at 
http://grants.nih.gov/grants/guide/notice-files/not98-030.html.

Submit a signed, typewritten, original of the application, including the 
checklist and five signed photocopies in one package to:

CENTER FOR SCIENTIFIC REVIEW
NATIONAL INSTITUTES OF HEALTH
6701 ROCKLEDGE DRIVE, ROOM 1040, MSC 7710
BETHESDA, MD 20892-7710
BETHESDA, MD 20817 (for express/courier service)

The title and number of the program announcement must be typed on line 2 of 
the face page of the application form and the YES box must be marked.

SPECIFIC APPLICATION INSTRUCTIONS FOR MODULAR GRANTS

The modular grant concept establishes specific modules in which direct costs 
may be requested as well as a maximum level for requested budgets. Only 
limited budgetary information is required under this approach. The just-in-
time concept allows applicants to submit certain information only when there 
is a possibility for an award. It is anticipated that these changes will 
reduce the administrative burden for the applicants, reviewers and Institute 
staff. The research grant application form PHS 398 (rev. 4/98) is to be used 
in applying for these grants, with the modifications noted below.

BUDGET INSTRUCTIONS

Modular Grant applications will request direct costs in $25,000 modules, up 
to a total direct cost request of $250,000 per year. (Applications that 
request more than $250,000 direct costs in any year must follow the 
traditional PHS398 application instructions.) The total direct costs must be 
requested in accordance with the program guidelines and the modifications 
made to the standard PHS 398 application instructions described below:

PHS 398

o  FACE PAGE:  Items 7a and 7b should be completed, indicating Direct Costs 
(in $25,000 increments up to a maximum of $250,000) and Total Costs [Modular 
Total Direct plus Facilities and Administrative (F&A) costs] for the initial 
budget period.  Items 8a and 8b should be completed indicating the Direct and 
Total Costs for the entire proposed period of support.

o  DETAILED BUDGET FOR THE INITIAL BUDGET PERIOD - Do not complete Form Page 
4 of the PHS 398.  It is not required and will not be accepted with the 
application.

o  BUDGET FOR THE ENTIRE PROPOSED PERIOD OF SUPPORT - Do not complete the 
categorical budget table on Form Page 5 of the PHS 398.  It is not required 
and will not be accepted with the application.

o  NARRATIVE BUDGET JUSTIFICATION - Prepare a Modular Grant Budget Narrative 
page. (See http://grants.nih.gov/grants/funding/modular/modular.htm for 
sample pages.)  At the top of the page, enter the total direct costs 
requested for each year. This is not a Form page.

o  Under Personnel, list key project personnel, including their names, 
percent of effort, and roles on the project. No individual salary information 
should be provided. However, the applicant should use the NIH appropriation 
language salary cap and the NIH policy for graduate student compensation in 
developing the budget request.

For Consortium/Contractual costs, provide an estimate of total costs (direct 
plus facilities and administrative) for each year, each rounded to the 
nearest $1,000.  List the individuals/ organizations with whom consortium or 
contractual arrangements have been made, the percent effort of key personnel, 
and the role on the project. Indicate whether the collaborating institution 
is foreign or domestic. The total cost for a  consortium/contractual 
arrangement is included in the overall requested modular direct cost amount. 
Include the Letter of Intent to establish a consortium.

o  BIOGRAPHICAL SKETCH - The Biographical Sketch provides information used by 
reviewers in the assessment of each individual's qualifications for a 
specific role in the proposed project, as well as to evaluate the overall 
qualifications of the research team.  A biographical sketch is required for 
all key personnel, following the instructions below.  No more than three 
pages may be used for each person.  A sample biographical sketch may be 
viewed at: http://grants.nih.gov/grants/funding/modular/modular.htm.

- Complete the educational block at the top of the form page;
- List position(s) and any honors;
- Provide information, including overall goals and responsibilities, on 
research projects ongoing or completed during the last three years;
- List selected peer-reviewed publications, with full citations.

o CHECKLIST - This page should be completed and submitted with the 
application.  If the F&A rate agreement has been established, indicate the 
type of agreement and the date. All appropriate exclusions must be applied in 
the calculation of the F&A costs for the initial budget period and all future 
budget years.

o The applicant should provide the name and phone number of the individual to 
contact concerning fiscal and administrative issues if additional information 
is necessary following the initial review.

REVIEW CONSIDERATIONS

Applications will be assigned on the basis of established Public Health 
Service referral guidelines.  Applications that are complete will be 
evaluated for scientific and technical merit by an appropriate peer review 
group convened in accordance with NIH peer review procedures. As part of the 
initial merit review, all applications will receive a written critique and 
undergo a process in which only those applications deemed to have the highest 
scientific merit, generally the top half of applications under review, will 
be discussed, assigned a priority score, and receive a second level review by 
the appropriate national advisory council or board.

The review criteria below are required for unsolicited research project grant 
applications. To the extent reasonable they are also to be used for other 
mechanisms. 

Review Criteria

The goals of NIH-supported research are to advance our understanding of 
biological  systems, improve the control of disease, and enhance health. In 
the written comments  reviewers will be asked to discuss the following 
aspects of the application in order to  judge the likelihood that the 
proposed research will have a substantial impact on the  pursuit of these 
goals. Each of these criteria will be addressed and considered in assigning 
the overall score, weighting them as appropriate for each application. Note 
that the application does not need to be strong in all categories to be 
judged likely to have major scientific impact and thus deserve a high 
priority score. For example, an investigator may propose to carry out 
important work that by its nature is not innovative,  but is essential to 
move a field forward.

1.  Significance:  Does this study address an important problem?  If the aims 
of the application are achieved, how will scientific knowledge be advanced?  
What will be the effect of these studies on the concepts or methods that 
drive this field?

2.  Approach:  Are the conceptual framework, design, methods, and analyses 
adequately developed, well-integrated, and appropriate to the aims of the 
project?  Does the applicant acknowledge potential problem areas and consider 
alternative tactics?

3.  Innovation:  Does the project employ novel concepts, approaches or 
method? Are the aims original and innovative?  Does the project challenge 
existing paradigms or develop new methodologies or technologies?

4.  Investigator:  Is the investigator appropriately trained and well suited 
to carry out this work?  Is the work proposed appropriate to the experience 
level of the principal investigator and other researchers (if any)?

5.  Environment:  Does the scientific environment in which the work will be 
done contribute to the probability of success?  Do the proposed experiments 
take advantage of unique features of the scientific environment or employ 
useful collaborative arrangements? Is there evidence of institutional 
support?

In addition to the above criteria, in accordance with NIH policy, all 
applications will also be reviewed with respect to the following:

o  The adequacy of plans to include both genders, minorities, and their 
subgroups as appropriate for the scientific goals of the research.  Plans for 
the recruitment and retention of subjects will also be evaluated.

o  The reasonableness of the proposed budget and duration in relation to the 
proposed research.

o  The adequacy of the proposed protection for humans, animals or the 
environment, to the extent they may be adversely affected by the project 
proposed in the application.

AWARD CRITERIA

Applications will compete for available funds with all other approved 
applications.  The following will be considered in making funding decisions:

o  Quality of the proposed project as determined by peer review
o  Availability of funds
o  Program priority.

INQUIRIES

Inquiries are encouraged. The opportunity to clarify any issues or questions 
from potential applicants is welcome.

Direct inquiries regarding programmatic issues to:

Huber R. Warner, Ph.D.
Biology of Aging Program
National Institute on Aging
7201 Wisconsin Avenue
Suite 2C231 - MSC 9205
Bethesda, MD 20892-9205
Telephone:  (301) 496-6402
FAX:  (301) 402-0010
Email:  warnerh@exmur.nia.nih.gov

or

Bradley Wise, Ph.D.
Neuroscience and Neuropsychology of Aging Program
National Institute on Aging
7201 Wisconsin Avenue
Suite 3C307 - MSC 9205
Bethesda, MD 20892-9205
Telephone:  (301) 496-9350
FAX:  (301) 496-1494
Email:  wiseb@exmur.nia.nih.gov

Direct inquiries regarding fiscal matters to:

Crystal Ferguson
Grants and Contracts Management Office
National Institute on Aging
7201 Wisconsin Avenue
Suite 2N212 - MSC 9205
Bethesda, MD 20892-9205
Telephone:  (301) 496-1472
FAX:  (301) 402-3672
Email:  fergusoc@exmur.nia.nih.gov

AUTHORITY AND REGULATIONS

This program is described in the Catalog of Federal Domestic Assistance No. 
93.866. Awards are made under authorization of sections 301 and 405 of the 
Public Health Service Act as amended (42 USC 241 and 284) and administered 
under NIH grants policies and Federal Regulations 42 CFR 52 and 45 CFR Parts 
74 and 92. This program is not subject to the intergovernmental review 
requirements of Executive Order 12372 or Health Systems Agency review.

The PHS strongly encourages all grant and contract recipients to provide a 
smoke-free workplace and promote the non-use of all tobacco products.  In 
addition, Public Law 103-227, the Pro-Children Act of 1994, prohibits smoking 
in certain facilities (or in some cases, any portion of a facility) in which 
regular or routine education, library, day care, health care or early 
childhood development services are provided to children.  This is consistent 
with the PHS mission to protect and advance the physical and mental health of 
the American people.

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