During the past FYs, several labs independently validated neuroprotection by p5 peptide after systemic i.p. injections or after AAV-9 delivery in animal models of PD and AD, respectively. In addition, p5 was shown to reduce the infarct size in an ischemic stroke model. These results demonstrate peptide efficacy and safety at least during short term applications, but they don't address potential long-term safety issues. The safety and efficacy of long-term systemic use of the peptide or focal peptide expression is critically important before any clinical use should even be considered. Potential detrimental effects of p5 on normal, synaptic Cdk5/p25 activity, which plays a role in LTD and neuroplasticity, must be ruled out. Evidence suggests that there are two types of Cdk5/p25 activities in neurons: normal synaptic and hyperactive cytoplasmic. Recent evidence does suggest that p5 selectively can reduce aberrant cytoplasmic Cdk5/p25 hyperactivity without interfering with synaptic plasticity. LV vectors created by our proposed project could directly be used to generate transgenic mice that express the secreted and cell-penetrating version of the peptide in most cells. This is a very important step to verify safety. The development of healthy transgenic mice, which express this new p5 peptide in every cell, behave normally and show normal cognition and motor functions, would be strong evidence for the safety of the peptide. Transgenic mice are an elegant solution to address key safety issues, while allowing to verify therapeutic efficacy in various animal disease models. It is anticipated that these mice resist neurodegeneration and ameliorate associated immune responses. The p5 fragment of p35 is highly conserved in most lab animals and in human, and obtained results will be relevant across species. Before extensive efficacy tests with the peptide are started, it would be more cost-effective and less labor-intensive to first generate these transgenic p5 mice to verify safety. Recent developments in non-toxic and non-immunocompromising conditioning regimens in mice, prior to hematopoietic stem cell transplantation, have shown great progress in other labs. In addition, new methods for the generation of iPSCs with their ability to develop into all lymphoid and myeloid cell types are very encouraging. It can be expected that transplantation procedures even for older AD and PD patients will become increasingly safe. These advances will allow safe and efficient targeted transgene delivery to the human CNS in the future, which could target sites with engaged immunological responses during wide-spread CNS neurodegeneration processes. Last FY, findings by an intramural lab at NINDS and by several outside labs continue to strongly support the notion that the p5 peptide can override toxic Cdk5/p25 hyperactivity in neurons and prevent neurodegeneration, as well as reduce associated inflammation on a cellular and systemic scale. In addition and critically important, neuroprotective i.p. injections of the cell-penetrating peptide in mice did not interfere with the neurological processes of memory and behavior. These studies are strong evidence for a continued favorable safety profile of p5 in mice, at least during relative short treatment periods. It suggests a safe therapeutic/protection potential during acute neuronal pathogenesis after stroke or traumatic brain injury. However, as outlined above, before advancing to clinical studies, an even more careful and detailed safety profile of the p5 peptide during long-term treatment is required. Outside the brain, Cdk5/p35 kinase participates in many additional physiological and pathological functions involved in cancer, senescence, inflammation, diabetes muscular dystrophy etc. It is essential that potential side effects of p5 on as yet unknown or less studied mechanisms be examined during long term treatments. Generating transgenic mice expressing a secreted cell-penetrating version of p5 in most cells together with its extracellular delivery to even more cells could serve as a more stringent test to study potential adverse effects from embryonic development until adulthood. The use of transgenic mice neatly bypasses the repeated 3-times weekly high levels of i.p.peptide injections. Co-expression of EGFP will identify peptide secreting cells. These mice may also be resistant to pathogenic secondary inflammatory responses after stroke or TBI. P5-expressing mice may be able to transfer their potential neurodegeneration-resistance either through breeding with established mouse lines of neurodegenerative disease for AD, ALS, or PD, or by the transfer of hematopoietic stem cells or other transplantable cell types derived from these transgenic mice. It is also important to appreciate that all of these studies will simultaneously address several challenges of effective gene/gene product delivery to the nervous system. Many aspects are key to safety and efficacy, but are rarely discussed. They include the sizes of fluid-filled interstitial spaces of the brain parenchyma and the CSF within perivascular and subarachnoid spaces, the amounts of circulating CSF, its flow kinetics and continual replacement. These parameters clearly affect therapeutics delivery especially for secreted therapeutics. While the p5 peptide serves as an excellent model to answer some of these important questions, fortunately, it is also an attractive candidate as a therapeutic agent.