Validation of neuroprotective effect of blackberries
Validation of neuroprotective effect of blackberries digested metabolites in a model of neurodegeneration based on rat neurons primary culture
CLOSED
Background
Recent consumer's interest in berries is due to their highly advertised health impact. Evidences include protection in neurological diseases, in particular during aging. The present proposal envisages to establish a neurodegeneration cell model based on neurons primary culture and to validate protective molecular mechanisms underlied by blackberry digested metabolites. In previous work performed in the lab it was demonstrated that blackberries, in particular Portuguese wild blackberry species could efficiently protect a neuroblastoma cell line from oxidative injury (1). In this work blackberries were in vitro digested since phytochemicals are greatly altered during gastrointestinal digestion. Also a physiological approach was considered by using serum relevant concentrations, that are reduced comparing to the amounts ingested (2). Accordingly to this, hardly phytochemicals could act in the brain as antioxidants per se, they are more prone to act trough specific biological targets. After establishment of the neurodegeneration model based on primary culture of neurons, berries digested metabolite effects on cell viability/ cell death mechanism and mitochondria biogenesis will be assessed.
Working plan
Task 1- Establishment of primary culture of neurons
A primary culture of neurons isolated form rodent (or from rats or mice) will be used for validation of the neuroprotective effect induced by digested metabolites previsouly detected on a neuroblastoma cell model (1). Primary culture of neurons will be isolated from 7 day old mice cerebellum accordingly to routine procedures (3).
Task 2- Establishment of neurodegeneration model using primary culture of neurons
A model consisting in challenging neurons primary culture with hydrogen peroxide, which mimics cellular oxidative stress and decreases cell viability will be established.
Task 3- Evaluation of neuroprotective effect of blackberries digested metabolites
The previously detected neuroprotective effect of berries metabolites will be confirmed in a more representative model, established in Task 2. The neuroprotection will be accessed by fluorescence microscopy using propidium iodide (PI) (1) and Hoechst and immunocytochemistry methodologies.
Task 4- Assessment of mitochondria biogenesis
Possible changes in mitochondrial population are important feature to be assessed in a neurodegeneration model induced by an oxidative insult. Thus, identifying whether digested metabolites induce mitochondrial biogenesis and how it may alter cellular metabolism and enforce oxidative phosphorylation is an important issue. Mitochondrial biogenesis will be assessed by measuring mitochondrial DNA (quantitative PCR), complex I-IV expressions (Western blots) and complex IV activity (enzymatic assays) (4).
References
1. Tavares, L., Figueira, I., McDougall, G., Vieira, H., Stewart, D., Alves, P., Ferreira, R., and Santos, C. (2013) European Journal of Nutrition 52, 225-236
2. Manach, C., Scalbert, A., Morand, C., Remesy, C., and Jimenez, L. (2004) The American Journal of Clinical Nutrition 79, 727-747
3. Vieira, H. L. A., Queiroga, C. S. F., and Alves, P. M. (2008) Journal of Neurochemistry 107, 375-384
4. Almeida, A. S., Queiroga, C. S. F., Sousa, M. F. Q., Alves, P. M., and Vieira, H. L. A. (2012) Journal of Biological Chemistry 287, 10761-10770
The present project is aligned with the aim of European project FP7-KBBE-2010-4 (265942) EUBerry.
Supervisors
Cláudia Nunes dos Santos (csantos@itqb.unl.pt)
Lucélia Rodrigues Tavares (ltavares@itqb.unl.pt)
Helena Vieira (helena.vieira@fcm.unl.pt)
Laboratory/Institution
