Progress 08/15/16 to 04/14/17

Outputs
Target Audience:A persistent, spreading inflammatory and oxidative stress (IOS) is largely responsible for the progressive degeneration ofneuronal connectivity reflected ultimately by a debilitating decline in cognitive function and accompanies most age and traumarelatedailments including Alzheimer's disease (AD), its early pathology Mild Cognitive Impairment (MCI), other chronicneurodegenerative CNS disorders (Parkinson's, ALS), acute CNS trauma (stroke, closed TBI), some complex brain disorders(Schizophrenia, bipolar disorders), and normal CNS aging. For most of these ailments, there is no cure or even a treatment forslowing their progression and age/lifestyle are the main risk factors. Presently, the yearly economic burden due to age-relatedcognitive decline is estimated to exceed $600 billion worldwide and about $200 billion in the US alone. With regard to AD pathology, an estimated one in eight adults 65 years or older (54 million) suffer from cognitive impairment, which is the 6 th leading cause of death in the US and the leading cause of dementia worldwide. These projections are even more concerning with the rapid increase in the aging population. Measures to blunt, delay or confine IOS alleviate or even halt neuronal degeneration and cognitive decline as shown in cellular and animal models. Cereon Biotechnology LLC aspires is to develop a functional foodwith GRAS designation, which specifically blunts the detrimental outcome of IOS on neurodegeneration. Lowering the IOS burden and strengthening synaptic integrity in the aging and diseased central nervous system could intervene with the progressive cognitive decline in the CNS and concomitantly stabilizing/promoting cognitive performance. This product would combine natural ingredients, target specificity, affordability, addressing an unmet need, and serve specifically an increasing segment of the US - the elderly population (55 years or older). In particular, individuals suffering frommild cognitive impairment, early Alzheimer's disease and potenially other neurodegenerative disease could benefit from this product. A functional food would be superior to current strategies, which largely act as choline esterase antagonist. These treatments offer limited efficacy and do not significantly slow progressive decline of cognitive capacities. High tolerability of functional foods could favor both preventative application as well as early intervention applications. The most optimal end point of utilizing this functional food would be a complete restoration of cognitive capacities and elimination of IOS. Changes/Problems:No major changes to the approach have occurred. Harvest parameters such as locations across the state of Alaska were less than planned due to a significant delay in the peak season of berries. Regardless locations from the far north (Wiseman) across the interior (Fairbanks and Gulkana) to the coastspanning over 5 degrees in lattitude and longitude were included in the study. Changes in peak berry season also negated the collection of berries in the early season for analysis. What opportunities for training and professional development has the project provided?The project has not directly provided training and/or professional development opportunities. Expansion of this project to a commercialization of this bioactivity will however generate ample opportunities across the STate of Alaska. Key areas would include harvesting and initial processing of berry materials, further processing to the final material, and concomitant quality testing of the process. Opportunities for individuals with expertise ranging from plant biology/horticulture, to biochemistry, to business managment will be developed. How have the results been disseminated to communities of interest?Informal information has been provided to individuals in the Fairbanks and Anchorage region on the activities of Cereon Biotechnology including invidiuals from the business communities. In addition, teaching activites of the PI on this research at the Osher Life Long Learning Center in Fairbanks provided ample opportunity to disseminate the activities of Cereon Biotechnology and more importantly the role of nutrition and botanical in the context of this research. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Objective 1 accomplishments: Non Polar fractions were obtained by solid phase chromatography (silica) and equated to about 6% of total dry mass of the original crude extacts. Alaska blueberries were harvested in Fairbanks, Wiseman, Anchorage, and Gulkana river basin as well as crow berries and lingon berries. For parity analysis, wild blueberries from Maine and commercial blueberries from Oregon were also processed. Non polar fractions were analyzed for their potency to quench ROS production by NADPH oxidase, to block assembly of the NADPH oxidase mutliprotein complex, lipid raft coalescence, neuronal viability, antioxidant capacity, and inhibiton of actin/cofilin rod-like inclusion bodies in cortical neurons. Non polar fractions from AK blueberries (all locations) exhibited potency as low as theng/ml range as well as non polar fractions from crow berries with respect to ROS quenching, bocking NOX assembly, blunting raft coalescence, and inhibiting rod formation. A rapid purification scheme was developed that will allow processing of larger quantities more effeciently - these non polar fractions exhibited activity in the low ng/ml range (< 200 ng/ml) as demonstrated in dose response assays. All crude berry extracts were capable to quench ROS formation including berries from Maine or Oregon. Antioxidant scavening activities of all non polar fractions was neglible and at concentrations used for curde extracts were below 0.05 mM Trolox equivalents. Noteably, Maine bluerries exhibit detectable potency against NOX assembly which was however over 6 fold less per dose response analysis compared to Alaska blueberry non polar fractions. Objective 2 accomplisments: Wild bog blueberries, lingon berries, and crow berries were collected in Wiseman (67o24'N - 150 o6'W), Fairbanks (64o50'N - 147 o43'W), and Gakona (62o18'N- 145 o16'W) (Interior regions) and the costal vicinity of Anchorage (61o13'N - 149 o 54'W) spanning over 5o in latitude (approximately 650 miles north-south) both during early season and peak season. Berries were kept frozen (-80oC) until processing. For parity analysis, we included commercial blueberries from Maine and Oregon. Berries from other costal regions were not available due to a delaly of peak season by more than 3 weeks. However, the assay systems targeting neuronal parameters crucial to inflammatory and oxidative stress-dependent neurodegeneration demonstrated that berries from all locatioins across the State of Alaska were equally potent. Importantly, extracts and non polar fractions from blueberries from Maine or Oregon were either significantly less potent (Maine) or not at all (Oregon). Objective 3 accomplishments: A rapid fractionation procedure was developed that enriches the bioactivity in Alaska blueberries to a similar extent as the solid phase chromatography procedure. The rapid fraction procedure is considered intelletual property (no details disclosed). It is significant that the rapid procedure will enable the processing of large quantities of berry materials. In summary, research activities have accompolished all major aspects set forward in the objetives and establish the feasibility to extend this research toward commercialization.

Publications