The influence of dimethyl fumarate on the spatial memory, neurogenesis, neurodegeneration and cerebral imflammation in the rat model of Alzheimer's disease

1. Research project objectives/ Research hypothesis

Animal models are very useful in studies on the mechanism and therapy of Alzheimer’s disease (AD). Transgenic animals with mutation of genes related to β-amyloid production are an accurate model of rare, early-onset, familiar-form AD. Prevailing, late-onset, sporadic-form AD (sAD) is related mostly to environmental factors. Decrease of glucose utilization in the cortex and hippocampus, as well as lower expression of insulin receptor and insulin-like growth factors were found in the brains of sAD patients. This “insulin-resistant brain state” and cognitive disorder accompanying AD is accurately reflected in the animal model of AD evoked by intracerebroventricular (i.c.v.) injection of betacytotoxic drug, streptozotocin (STZ). This model of sAD reflects also cholinergic deficit, activation of microglia and oxidative stress-related neurodegeneration. These symptoms lead finally to tau protein and β-amyloid expression. This project aims to determine if dimethyl fumarate (DMF), which has antioxidative and immunosuppressive properties, when taken orally for three weeks, can alleviate spatial working memory impairments, cerebral inflammatory processes, and neurodegeneration, and improve neurogenesis in the STZ-induced rat model of AD. DMF will be tested in the animal model of AD for the first time. DMF was recently tested clinically on patients with another neurodegenerative disease: multiple sclerosis. It induces the transcriptional factor Nrf2 (nuclear factor [erythroid-derived 2]-related factor 2), which activates the expression of several antioxidant enzymes. It also shows anti-inflammatory properties by reduction of secretion of inflammatory mediators by activated microglia in vitro. This project will test the hypothesis that DMF can reduce the expression of pro-inflammatory cytokines in the rat brain.The influence of age and individual stress-reactivity on the action of DMF in the rat model of AD will also be tested.

After i.c.v injections of STZ the memory deficit correlates with age, and in older animals interaction of cognitive impairment and cholinergic deficit was found. Animals will be divided into behavioural groups according to their locomotor response to novelty correlating with the reactivity of the hypothalamic-pituitary-adrenal axis (PPN). High responders to novelty (HRs), due to higher reactivity of the PPN axis, are considered to be sensitive to stress. Aged HRs are more vulnerable to memory deficits compared to aged low responders to novelty (LRs), which is an effect of long-term increase in corticosterone level influencing hippocampal neurogenesis and plasticity.

2. Research project methodology

Four-month-old rats divided into HRs (n=88) and LRs (n=88) will be allocated randomly to two main groups: YOUNG (immediately subjected to further procedures, 40 HRs and 40 LRs) OLD (subjected to further procedures at the age of 22 months; 48 HRs and 48 LRs). Higher numbers in the OLD group will compensate for mortality related to age. Out of each main group, four subgroups will be separated (n = 10 HRs + 10 LRs in YOUNG or 12 HRs + 12 LRs in OLD group):

1. STZ DMF subjected to i.c.v. injection of STZ, fed with 0.4% DMF fodder

2. VEH DMF subjected to i.c.v. injection of vehicle, fed with 0.4% DMF fodder

3. STZ CTR subjected to i.c.v. injection of STZ, fed with standard fodder

4. VEH CTR subjected to i.c.v. injection of vehicle, fed with standard fodder

The following parameters will be measured:

1) spatial reference and working memory (in Morris water maze)

2) number of basal forebrain cholinergic neurons (choline acetyltransferase [ChAT] immunohistochemistry)

3) level of hippocampal neurogenesis (the double immunofluorescent labelling of 5-bromo-2’deoksyurydine [BrDU, mitotic cells marker] and the doublecortin [DCX, immature neurons marker])

4) level of neurodegeneration (with the use of Fluoro-Jade B, marker or necrotic/apoptotic neurons)

5) number of activated microglia (immunohistochemistry anti-CD68)

6) brain expression of cytokines: pro-inflammatory IL-6 or anti-inflammatory IL-10 and brain-derived neurotrophic factor (BDNF) (double immunofluorescent dyeing: IL6 + BDNF, IL10 + BDNF)

3. Expected impact of the research project on the development of science, civilization and society

Confirmation of the efficacy of DMF in alleviation of cognitive deficit, neurodegeneration and cerebral inflammation in the rat model of sAD would initiate further research on other models of AD in animals and even clinical trials of DMF as a supporting drug for AD patients. This project will also provide information on the influence of the age factor and individual vulnerability, stressing the possible therapeutic action of DMF.