RUOLO PREDITTIVO DELL'ESPRESSIONE DEL RECETTORE SCAVENGER CD36 NELLA MALATTIA DI ALZHEIMER

Among pathologies of the old people, dementia represent one of the main sanitary problems. Approximately 70% of the dementia cases it is represented by the Alzheimer’s Disease, a neurodegenerative cerebral pathology, whose prevalence is supposed to be increased in the next years. One pathological hallmark is represented by amyloid-beta (Aβ) plaques, which represent the key element to oxidative and pro-inflammatory mechanisms by the activated microglia. The interaction, in fact, between activated microglia and plaques of Aβ induces the release of pro-inflammatory citokynes, like interleukin-6 (IL-6), or neurotoxic factors, like the tumor necrosis factor-α, and stimulates the expression by the microglia of some scavengers receptors, deputies to remove the Aβ plaques: among them the multifunctional protein of class B type I (SRBI) CD36. Currently an effective diagnosis of AD is possible only post mortem: for this reason during the last few years has been intensified the search for biological and hormonal markers that could be useful in the early diagnosis of AD. These markers are expressed also from the peripheral leucocytes, cells easily obtainable, which express virtually all hormones and hormone receptors, which are under the same regulatory mechanisms that control their expression in the brain. So the leucocytes may be profitably used as tools to investigated the changes occurring in brain areas reportedly inaccessible in humans. In particular, it has been recently demonstrated that the leukocyte expression of CD36 is significantly reduced in patients with AD and in patients with mild cognitive impairment (MCI), a prodromic phase of AD. CD36 could represent an earlier marker of increased neurodegenerative risk. Epidemiologic studies have shown a greater incidence of AD in the individuals of female sex. A possible cause of this is represented by the modifications of the endocrine functions, that occur during the menopausal transition and that develop an unfavorable hormonal milieu predisposing to the neurodegeneration. It is demonstrated that estrogens have a neuroprotective and neurotrophic role by the binding to specific receptors, present in two isoformes (ER-α and ER-β), and that in course of AD they prevent the formation of the Aβ-fibrils, inhibit the inflammatory reaction due to the plaque deposition, and protect the cells from their cytotoxic action. The diminished estrogen secretion in postmenopausal age, in fact, is correlated inversely with the “cerebral health” and directly with the entity of the cognitive deterioration. An other female sexual hormone, the progesterone, seems to carry out multiple functions centers different from the riproduction function: the regulation of the cognitive processes, the mitochondrial function of the neuronal cell, the neurogenesis and the repair of the damaged nervous tissue. The neuronal response regulated from the progesterone are mediated by the receptors PR-A and PR-B, included some forms derived from alternative splicing. At the level of the central nervous system them (CNS), therefore, the estrogens and the progesterone cooperate in regulating some neuronal functions, such as the neuroprotection and various cognitive processes. The PR gene contains promoter sequences able to bind ER; therefore, the gene expression of PR supposes the existence of one (efficient) estrogenic stimulation. Moreover, it has been demonstrated the existence of points of convergence between intracellular signaling of estrogens and the factor of insulin-like growth factor type 1 (IGF-1), a neuromodulator that regulates the synaptic plasticity and has been involved in the tissue regeneration, guaranteeing protection during the neurodegenerative processes. Based on this, in the present thesis we evaluated the leukocyte expression of some biological parameters, in order to clearly understand the balance between the neuroprotective and proneurotoxic hormonal factors in during the life, focusing the interest on the phase of menopausal transition, a critical period of the women life during which conditions predisposing to the neurodegeneration occur. We recruited, health male and female subjects as control, of age between 20 and 91 years, and AD patients, both male and female. Fasting blood samples were drawn from all the subjects to isolated the leucocytes and the plasma. With RT-PCR we analyzed the CD36 expression, ER-α, ER-β, PR-A/B, IGF-R1 and IL-6 on the leucocytes and, by RIA or ELISA, the levels of estradiol, progesterone, IGF-1 and IL-6 in the plasma, with the following results: • as aspected, the leukocyte expression of CD36 was significantly reduced in the AD patients than the controls, young and old. Moreover, while in the healthy males the expression was not modified with the age, in the women it endured a remarkable reduction in the period correspondent to the menopausal transition (decade of age between 51 and 60 years), in order then to return to normal levels in the older subjects; interesting was that in women with AD the values were similar to those found in the women in peri-menopause; • the leukocyte expression of ER-α and ER-β in the women of 51 and 60 years of age was significantly increased, while in the men was quitly constant in the course of the life and in the AD patients of both sexes it wasn’t different by the healthy subjects of comparable age; • the leukocyte expression of PR-A/B in the healthy men was lower than the women of the same age and progressively it increased until a peak in the decade of age between 41 and 50 years, after that it was reduced in the successive decades in order then to remain stable with increasing of the age. In the women, instead, there was two-phase pathway with a minimum in the subjects 41-50 years old and higher values in older and younger. In the patients with AD of both sexes the PR-A/B expression was similar to that observed in control subjects; • the leukocyte expression of IGF-1R in the male controls diminished with increasing of the age, while in the women this decrease was less evident. In the AD patients of male sex the IGF-1R expression was significantly higher of that of the healthy subjects of the same sex; • the leukocyte expression of IL-6 in the male control population was constant while in the female population occurs an abrupt increase in the decade between 51 and 60 years. In the AD patients of both sexes the IL-6 expression was lower than the healthy controls of comparable age; • the plasmatic levels of estradiol and progesterone in the healthy men did not vary according to the age. On the contrary, in the women starting with the menopause the both hormones levels were significantly reduced. In the AD men the levels of estradiol0 were lower than the controls, while in the AD women they were similar to those found in the controls of comparable age; • the plasmatic levels of IGF-1 progressively diminished in the healthy subjects of both sexes. This reduction was not only more evident, but also more precocious in women than men. The levels of IGF-1 were significantly higher in the AD patients of both sexes than the control subjects of equal age; • none of the biological parameters investigated was related to age, except for the estradiol and progesterone levels in women and IGF-1 in both sexes; • in the healthy population of both sexes a direct correlation between the expression of ER-α or ER-β and IL-6 and between the concentrations of IGF-1 and the expression of IGF-1R was observed. In order to estimate the role of IGF-1 in the menopausal transition, the female pre- and post- menopausal population has been subdivided in the groups with normal or reduced levels of IGF-1 based on a predefined cut-off: in the group of pre-menopause women with normal levels of IGF-1 the plasma concentrations of estradiol were positively related to the leukocyte expression of PR-A/B, but this association was less evident when the group with reduced levels of IGF-1 was considered. On the contrary, in post-menopause women with normal or reduced levels of IGF-1 any statistically significative correlation between the two parameters was observed. On the base of these data it can be asserted that the phase of menopausal transition represents a “critical temporal window” in which an hormonal milieu characterized by a strong loss of balance of various hormonal factors and by the evident prevalence of pro-neurotoxic infuences is developed (in particular, the reduction of the estrogen protection, which in presence of normal levels of IGF-1, is not able to stimulating the PR-A/B expression and to reduce the production of IL-6). This could be a favoring element to develop neurodegeneration. In the second part of this thesis we decided to investigated the effectiveness of estrogens in modifying, in favorable sense, the balance between neuroprotective vs. pro-neurotoxic infuences (for eg., reduction of microglial expression of CD36 by citokynes stimulation) in vitro For this study we treated murine microglial immortalized N9 cells with 17--estradiol and TNF- in a time-dependent way: estradiol before, after or simultaneously to TNF-, in order to analize the effect of a precocious, late or simultaneous 17--estradiol administration on the CD36 expression. As previously said, the CD36 reduction is a phenomenon predisposing to the disease; in fact TNF-α, one of the citochine mainly expressed in course of AD, induce a significative reduction of CD36. Only a precocious treatment with 17-β-estradiolo was able to revert the effect of the pro-inflammatory citokyne, demonstrating the effective existence of a temporal window in which the estrogen carries out its protecting role. Moreover, since is increased the tendency to the prescription of isoflavones derived from soy (phytoestrogens) in order to contrast the hot flushes, which plague many women in the precocious menopause phases, could be useful to investigate the possiblel neuroprotective ability to these compounds, evaluating the effects of hormonal replacement therapy (HRT) or of a therapy with phytoestrogens on the expression of CD36, as precocious biochemical marker of increased dementia risk by Real-Time PCR, or by mmunostaining For this study we used females rats, subdivided into 4 groups: intact + vehicle, ovariectomized + vehicle, ovariectomized + 17-β-estradiol and ovariectomied + phytoestrogen and we investigated some parameters of correct estrogenic replacement, with the following result: • analysis of the body weight: it was higher in the ovariectomied animals, and in the animals in phytoestrogen therapy, while the intact animals and those in therapy with estrogen were less fat, although the weights of the two groups of animals were not exactly the same; • analysis of the uterus weight: the ovariectomy and the phytoestrogen therapy induced the uterus atrophy, while the intact animals or in therapy with estrogens showed trophic uteri, although with a discrepancy between the two groups; • analysis of the femoral bone denity: the ovariectomy and the phytoestrogen therapy reduced the femoral mineral bone density (diaphisis and metaphisis included), while the intact animals and those in therapy with estrogen, the bone density was not affected and the values of bone density of the two groups were very similar, except about diaphisis; • plasma levels of 17-β-estradiol: the levels were lower in the ovariectomized group and in the phytoestrogen therapy group, while they were higher, but always different, in the intact animals and those in therapy with estrogen; • cerebral gene expression of CD36 (PCR): CD36 expression in the ippocampus and in the cortex of ovariectomized animals was low and the therapy with 17-β-estradiol was able to revert this effect, bringing back it to the levels of the intact group; • peripheral gene expression of CD36: it did not vary between the 4 groups; • CNS protein expression of CD36 (immunostaining): it did not changes in the various groups and tended to form some clusters; • CNS protein expression of GFAP (immunostaining): the animals shown a state of gliosis age-related, but no any difference occurred between the several groups; • CNS protein expression of Ferritina (immunostaining): the staining was similar to that for CD36, except for the endoteliali cells. Analyzing the result about the body weight, the uterus, the bone and the plasma concentrations of 17-β-estradiol in these animals we can conclude that the estrogen exogen administration was efficient to contrast the ipoestrogenic assessment. Moreover, in considering the data obtained in human subjects, we can maintain a loss of balance during menopause towards unfavorable factors, as CD36 reduction. The discrepancy between gene and protein CD36 expression probably is due to a different method sensibility. At last, considering the role wide demonstrated in literature of CD36 in the pathogeneses of AD and to test the possibility to use CD36 as precocious biochemical marker of disease, we investigated the CD36 expression in APP23 mice, valid model of AD, by Real-Time PCR and immunostaining. To We used animals of 1,3,6,9 and 12 months of age, in which the deposition of plaques begins around the sixth month of age; at the same time we assist at the maximum gene and protein expression of CD36, in order then to return to lower levels. Moreover, a staining with tioflavina S, which stains for Aβ plaques, showed an inverse relation between plaque and CD36, determining for this receptor a role in the acute phase of disease. In conclusion, the protecting effect of estrogens in contrasting the CD36 reduction, is showed to be time and dose-dependent. Moreover, would seem to have a precocious in the diagnosis of this disease.