Neuroprotective CNS barriers in neurological diseases.

The goal of this Program Announcement with set-aside funds (PAS) is to invite applications for studying the neurobiological and cerebrovascular mechanisms through which the neuroprotective blood-brain and blood-csf barriers function in the healthy and diseased adult, aged and pediatric brain. Blood-Brain Barrier (BBB) research embodies the true meaning of a "translational model" of neuroscience wherein breakthroughs in basic neuroscience are delivered to the clinic and require an agent delivery strategy and/or the ability to target specific areas of the brain. This PAS encourages studies focused on improving our understanding of the neuroprotective CNS barriers and enhancing the effectiveness of drug and gene delivery strategies for treatment of neurological diseases. Chief among the challenges to be addressed is the need to increase our knowledge about the molecular and cellular biology, cells of origin, gene and protein expression, and the regional differences of brain microvascular endothelial cells and pericytes and their interactions with adjacent brain cells.

A major challenge for treatment of most brain disorders is overcoming the difficulty of delivering therapeutic agents to specific regions of the brain. In its neuroprotective role, the blood-brain barrier (BBB) functions to hinder the delivery of many potentially important diagnostic and therapeutic agents to the brain. Therapeutic molecules and genes that might otherwise be effective in diagnosis and therapy do not cross the BBB into the brain in adequate amounts. Improving our knowledge of the molecular and cellular biology of the brain microvasculature and their interactions with surrounding brain cells, which constitutes the BBB in vivo, could lead to innovative strategies for drug and gene targeting to injured or disease tissue. Also, research is needed on the role of the brain microvasculature in protecting the brain from toxic agents and how damage to the BBB leads to long-term neurological toxicity in the development of many neurological diseases.

Understanding the basic biology of how the BBB works under normal and disease conditions across the lifespan may also provide insight on the integrative function of the brain. Research focused on cerebrovascular endothelial cell biology may provide insight into the "neurovascular unit", a conceptual model that considers brain function from the perspective of interactions among blood cells, endothelium, glia, pericytes, extracellular matrix and neurons.

This initiative was identified as the top research priority of the Brain Tumor Progress Review Group (PRG). The Stroke Progress Review Group has also identified neurovascular research and BBB biology as a high priority for advancing our understanding of stroke and brain function. Both Progress Review Groups are responsive to Congressional requests for planning in these areas. Also, the National Institute of Neurological Disorders and Stroke (NINDS) Neuroscience at the New Millennium Plan clearly identifies research on the blood-brain barrier as a scientific priority. The scientific support for this initiative can be found in these reports on the NINDS homepage at http://www.ninds.nih.gov/funding/ neural_environment/index.htm#research

This PAS is intended to achieve a better understanding of the effects of neurological disorders on the blood-brain barrier (BBB), improve our knowledge of- BBB biology and how it may contribute to the initiation and/or progression of neurological disease over the lifespan and develop new approaches for targeting the BBB, based on biological considerations, in order to improve drug delivery and target treatment of one or more such disorders.

Applications that address gene and protein expression for microvascular endothelial cells within normal, aging, and diseased brain such as gliomas or the ischemic penumbra are encouraged. Cerebrovascular genomics is considered a high priority. Because only very abundant BBB-specific transcripts will be detected with whole-brain gene microarrays, cerebrovascular genomics research needs to start with the initial isolation of brain capillaries from animal or human brain, both normal and perturbed. Comparison of capillaries from normal brain and perturbed tissue can help to elucidate the tissue-specific gene expression. Pattern-specific tissue expression could provide the platform for further investigations on overall brain vascular biology as it pertains to conditions such as angiogenesis, cell adhesion, antigen presentation, metastasis, cell-cell communication and local inflammation.

There are several modalities for drug and gene delivery through the BBB. They include: BBB disruption; the use of endogenous transport systems, including carrier-mediated transporters such as glucose and amino acid carriers; receptor-mediated transcytosis, systems such as the insulin or transferrin receptor; and active efflux transporters such as p-glycoprotein and the associated anti-porters. Studies to determine which strategies are most effective and how they can be improved for patients with neurological diseases are encouraged. Research areas appropriate for this PAS include, but are not limited by the following examples: 1) Develop and characterize in vivo and in vitro models that reflect the unique features of the BBB as translational models of neurological disease. Existing in vivo models, such as those for stroke or lysosomal storage diseases, may also prove useful for studying the structure and dynamics of the BBB. Studies that validate in vitro results with in vivo models are encouraged; 2) Examination of the genes and proteins that are uniquely expressed by the intact BBB and mechanisms by which brain cells regulate endothelial cell gene expression. This includes changes that occur in response to neurological disease or the aging process, for example, characterization of molecular signatures for disease diagnosis and targeting; 3) Explore the genesis and regulation of the BBB, its stem cell origins and remodeling of the (diseased/damaged/ aged) brain microvasculature; 4) Identify signal transduction pathways of brain capillary endothelial transcytosis and tight junction regulation under normal and disease conditions; 5) Characterize endogenous influx and efflux properties of the barriers including transporters in luminal and abluminal membranes of brain endothelium and epithelium; 6) Identify regional diversity of barrier properties within the brain and spinal cord microvasculature; 7) Characterize brain endothelial tight junction proteins in normal and disease states; 8) Investigate the various enzymatic barrier mechanisms; 9)Characterize membrane protein expression by cells of the BBB; 10) Development of novel brain drug and gene delivery methods based on unique properties of the BBB including gene therapy via vectors or via modified autologous cell transfer; 11) Explore the molecular basis of microbial interactions with brain endothelium; 12) Develop neuroimaging tools to identify changes in BBB permeability in vivo; 13) Examination of the molecular and cellular mechanisms of leukocyte migration at the BBB throughout the lifespan; 14) Comparison of transport systems in brain endothelia with choroids plexus epithelium as well as systemic epithelial cells; 15) Explore the interactions among the cellular and matrix elements of the BBB, for example, the microvascular basement membrane; 16) Examine the plasticity of the blood-brain and blood-CSF interfaces throughout the lifespan.

Applications should focus on neurological disorders relevant to the research missions of NINDS, the National Institue of Mental Health (NIMH) and/or the National Institute on Aging (NIA). A partial list of diseases of interest to NINDS is given in Appendix A of the planning document Neuroscience at the New Millennium; see http://www.ninds.nih. gov/about_ninds/strategic_plan.htm.

These include neurological disorders (e.g. stroke, brain tumors, Parkinson's disease, brain and spinal cord trauma, epilepsy, multiple sclerosis, brain lysosomal storage disorders, neuro-AIDS and Alzheimer's disease). The NIMH is interested in mechanistic studies of trafficking of cells, immune molecules and drugs across the blood-brain and blood-csf barriers during development and adulthood and how these processes impact the pathogenesis of neuroAIDS and mental disorders; see http://www.nimh.nih.gov/research/nimh-webs.elm.

NIA is interested in age-related neurode-generative disorders, such as Alzheimer's disease, brain injury, and impairments in cognitive, motor and sensory functions. Research areas relevant to the mission of the NIA can be found at: http://www.nia.nih.gov/research/extramural/ neuroscience/programs.htm.

A large amount of basic research is needed to significantly change how we translate neuroscience research bidirectionally. This PAS is focused on stimulating new concepts in the BBB field through the exploratory/developmental grant (R21) and the R01 mechanisms. The current workforce in the BBB field is small relative to the scientific and clinical needs for improved understanding of the BBB and progress will require collaboration among current investigators and scientists from disciplines not currently working in this area. Therefore, training and early career development will bc encouraged (please contact Program Staff for additional information). This PAS will remain active for 3 years to address the many gaps in our knowledge of how the neuroprotective barriers function and the time needed to increase the workforce in this critically important translational research area.

As an applicant, you will be solely responsible for planning, directing, and executing the proposed project. Applicants are encouraged to contact program staff for advice about choosing the appropriate grant mechanism.

The R21 mechanism (see http:// grants.nih.gov/grants/guide/pa-files/PA-03107.html) is intended to encourage new exploratory/developmental research projects by providing support for the early stages of their development. For example, such projects could assess the feasibility of a novel area of investigation or a new experimental system that has the potential to enhance health-related research. These studies may involve considerable risk but may lead to a breakthrough in a particular area, or to the development of novel techniques, agents, methodologies, models or applications that could have major impact on a field of biomedical, behavioral, or clinical research.

Applications for R21 awards should describe projects distinct from those supported through the traditional R01 mechanism. For example, long-term projects, or projects designed to increase knowledge in a well-established area will not be considered for R21 awards. Applications submitted under this mechanism should be exploratory and novel. These studies should break new ground or extend previous discoveries toward new directions or applications. R21 applications may request a project period of up to two years with a combined budget for direct costs of up $275,000 for the two-year period. For example, you may request $100,000 in the first year and $175,000 in the second year. The request should be tailored to the needs of your project. Normally, no more than $200,000 may be requested in any single year.

This PAS uses just-in-time concepts. It also uses the modular budgeting as wen as the non-modular budgeting formats (see http://grants.nih.gov/grants/funding/modular/modular.htm). Specifically, if you are submitting an application with direct costs in each year of $250,000 or less, use the modular budget format. Otherwise follow the instructions for non-modular budget research grant applications. This program does not require cost sharing as defined in the current NIH Grants Policy Statement at http:// grants.nih.gov/grants/policy/nihgps_2001/ part_i_1.htm.

Competing continuation applications submitted in response to this PAS will compete with all investigator-initiated applications and be referred and reviewed according to the customary peer review procedures. Responsibility for the planning, direction, and execution of the proposed project will be solely that of the applicant. The earliest anticipated award date is June 1, 2004.

NINDS, NIMH, and NIA have set aside $2,000,000 in total costs per year, in addition to funds available for applications sent in response to this PA that score within the NINDS payline (see NINDS Funding Strategy at http://www.ninds.nih.gov/ funding/ninds_funding_strategy.htm), depending on the overall scientific merit of the applications and the availability of funds throughout the duration of this solicitation (3 years). Applications submitted in response to this PAS will compete with all investigator-initiated applications for funding.

The total project period for an application submitted in response to this PAS may not exceed 5 years. Because the nature and scope of the research proposed may vary, it is anticipated that the size of each award will also vary. Although the financial plans of the Institute provide support for this program, awards pursuant to this PAS are contingent upon the availability of funds and the receipt of a sufficient number of meritorious applications.

Applications must be prepared using the PHS 398 research grant application instructions and forms (rev. 5/2001). Applications must have a Dun and Bradstreet (D&B) Data Universal Numbering System (DUNS) number as the Universal Identifier when applying for Federal grants or cooperative agreements. The DUNS number can be obtained by calling [phone] or through the web site at http://www.dunandbradstreet.com. The DUNS number should be entered on line 11 of the face page of the PHS 398 form. The PHS 398 is available at http://grants.nih.gov/ grants/funding/phs398/phs398.html in an interactive format. For further assistance contact GrantsInfo, 301-435-0714, email: [e-mail].

Applications submitted in response to this PAS will be accepted at the standard application deadlines, which are available at http://grants.nih.gov/grants/dates.htm. Application deadlines are also indicated in the PHS 398 application kit. Please note that AIDS related applications have separate receipt dates.

Supplemental Instructions: All instructions for the PHS 398 (rev. 5/2001) must be followed, with these exceptions: 1) Research Plan. For R21 applications only, items a--d of the Research Plan (Specific Aims, Background and Significance, Preliminary Studies, and Research Design and Methods) may not exceed a total of 15 pages. No preliminary data is required for R21 proposals, but may be included if it is available. Please note that a Progress Report is not needed for R21 awards; competing continuation applications for an exploratory/developmental grant will not be accepted. 2) Appendix. Use the instructions for the appendix detailed in the PHS 398 except that for R21 applications, no more than 5 manuscripts, previously accepted for publication, may be included.

Applications must be received by or mailed on or before the receipt dates described at http://grants.nih.gov/grants/funding/submissionschedule.htm. The CSR will not accept any application in response to this PAS that is essentially the same as one currently pending initial review unless the applicant withdraws the pending application. The CSR will not accept any application that is essentially the same as one already reviewed. This does not preclude the submission of a substantial revision of an unfunded version of an application already reviewed, but such application must include an Introduction addressing the previous critique.

Contact: Thomas P. Jacobs, NINDS, Neuroscience Center, Rm 2112, 6001 Executive Blvd, Bethesda, MD 20892-9527 USA, 301-496-1431, fax: [phone], email: [e-mail]; Jeymohan Joseph, NIMH, Neuroscience Center, Rm 6202 6001 Executive Blvd, Bethesda, MD 20892-9527 USA, 301-443-3012, fax: [phone], email: [e-mail]; Bradley C. Wise, NIA, 7201 Wisconsin Avenue, Suite 350, Bethesda, MD 20892-9205 USA, 301-496-9350, fax: [phone], email: [e-mail]. Reference: PA No. PAS-03-165