Caracterització de les estructures granulars patològiques presents a l’hipocamp dels ratolins amb senescència accelerada SAMP8

Abstract

La soca de ratolins amb senescència accelerada SAMP8 és utilitzada habitualment com a model de senescència; i el seu ús per a l’estudi de la malaltia d’Alzheimer s’ha estès degut a la manifestació espontània dels seus trets histopatològics distintius. En estudis recents es va identificar la presència de grànuls amiloides a l’hipocamp dels ratolins SAMP8. Aquests clústers de grànuls augmenten amb l’edat i s’estenen al llarg de l’hipocamp. A més a més, les característiques morfològiques dels grànuls amiloides són molt similars als grànuls tenyits amb Periodic Acid Schiff (PAS), descrits anteriorment en els SAMP8 i en ratolins envellits d’altres soques. L’objectiu principal d’aquesta tesi ha estat estudiar la composició, l’origen i el desenvolupament dels grànuls que apareixen amb l’edat a l’hipocamp dels ratolins senescents SAMP8 amb la finalitat d’aportar nova informació sobre la neuropatologia d’aquests ratolins per al seu ús com a model de la malaltia d’Alzheimer. En aquest treball s’ha identificat la correspondència entre els grànuls amiloides i els grànuls PAS de l’hipocamp d’aquests animals. Aquests grànuls són el resultat d’un procés degeneratiu present en els astròcits i que pot afectar també zones properes, i estan formats per fragments membranosos provinents de l’acumulació de restes d’estructures i orgànuls, com els mitocondris. A més a més, s’ha identificat la presència d’un neo-epítop en el nucli dels grànuls que és, almenys parcialment, de naturalesa glucídica. Aquest neo-epítop és reconegut per anticossos IgM contaminants presents en anticossos comercials produïts en ascites de ratolí o sèrum de ratolí i conill. Aquestes IgM contaminants són probablement anticossos naturals, i algunes IgM hemaglutinen de forma positiva i específica els eritròcits humans de tipus A. La presència tant del neo-epítop com de les IgMs han estat la causa de falses tincions positives que han generat interpretacions errònies del significat fisiopatològic dels grànuls. És el cas, per exemple, de la presència de β-amiloide o la proteïna tau en aquestes estructures. Finalment, les lesions cerebrals de pacients associades a malalties neurodegeneratives i a l’envelliment no corresponen als clústers de grànuls de l’hipocamp d’aquests ratolins, tot i que seria interessant estudiar la presència del neo-epítop en aquestes condicions fisiopatològiques.

SAMP8 mice present an accelerated senescence and are usually used as a model of aging. Because this strain of mice spontaneously expresses several histopathological features of Alzheimer’s disease (AD), it has been recently used as a model for studying this disease. Alterations in the expression of β-amyloid (Aβ) peptides, tau hyperphosphorilation, an increase of oxidative stress and gliosis suggest that SAMP8 mice could be a relevant model for studying the first stages of sporadic AD and its relation to aging.

Recent studies in our group identified the presence of Aβ clustered granules in the hippocampus of SAMP8 mice when immunohistochemical stainings were performed with antibodies directed against Aβ peptides. These clustered granules increase in number and extension with age, and they spread throughout the hippocampus. The features of Aβ clustered granules are very similar to the granules stained with Periodic Acid Schiff (PAS) already described in SAMP8 mice, as well as in aged mice of other strains. On the other hand, human brain lesions present in aging and other neurodegenerative diseases, such as AD, may vary their chemical composition with time, suffering substantial alterations in the principal components and the addition of secondary components.

The main objective of this thesis is to study the composition, origin and development of the granules that appear with age in the hippocampus of SAMP8 mice, with the aim of further describing the neuropathology of these animals and provide new information for using them as a murine model of AD. Therefore, in this thesis we studied the composition and order of appearance of the constituents of the hippocampal granules of SAMP8 mice, and the possible coincidence between Aβ and PAS granules. The morphology, ultrastructure, cellular origin and formation process of these structures has been also ascertained; and a comparison between these structures and human brain lesions of neurodegenerative diseases have been performed.

The results obtained in this thesis allowed reaching several conclusions. The pathological clustered granules of the hippocampus of aged SAMP8 mice correspond to the PAS granules described in aged mice of several mouse strains. These granules are the result of a degenerative process located in astrocytes that could affect the surrounding neuropil. In the granules formation process, several abnormal membranous structures and cellular organelles, like mitochondria, produce membranous waste that finally accummulates forming the core of the granules. On the other hand, a neo-epitope absent before the granules formation appears in the membranous residues. This neo-epitope is located in the nucleus of the mature granules and contains at least some structures of glycosidic nature. The neo-epitope is recognized by contaminant IgM antibodies present in a wide range of commercial antibodies produced in mouse ascites or mouse and rabbit sera, which are probably natural antibodies, and some of these IgMs haemmagglutinate A-type human erythrocytes, but not B and O subtypes. The neo-epitope could be considered an A-like epitope because monoclonal antibodies anti-A-type human erythrocytes do not recognize it. Both the neo-epitope presence in the granular structures and the IgMs contained in commercial antibodies have been the cause of false-positive stainings, and as a result, they have generated misunderstandings about the composition and physiopathological significance of the hippocampal granules of aged mice, as is the case of Aβ peptides and tau protein, which are not contained in these granules. Finally, human brain lesions described to date in patients of neurodegenerative diseases or aged individuals do not correspond to the clustered pathological granules present in the hippocampus of aged mice. The study of the presence of the neo-epitope in diseased human brain could be interesting in the understanding of the neuropathology of aging and neurodegenerative diseases.