Antonia Gutierrez

Inflammatory Response in the Hippocampus of PS1M146L/APP751SL Mouse Model of Alzheimer's Disease: Age-Dependent Switch in the Microglial Phenotype from Alternative to Classic

Although the microglial activation is concomitant to the Alzheimers disease, its precise role (neuroprotection vs neurodegeneration) has not yet been resolved. Here, we show the existence of an age-dependent phenotypic change of microglial activation in the hippocampus of PS1xAPP model, from an alternative activation state with Aβ phagocytic capabilities (at 6 months) to a classic cytotoxic phenotype (expressing TNF-α and related factors) at 18 months of age. This switch was coincident with high levels of soluble Aβ oligomers and a significant pyramidal neurodegeneration. In vitro assays, using astromicroglial cultures, demonstrated that oligomeric Aβ42 and soluble extracts from 18-month-old PS1xAPP hippocampus produced a potent TNF-α induction whereas monomeric Aβ42 and soluble extract from 6- or 18-month-old control and 6-month-old PS1xAPP hippocampi produced no stimulation. This stimulatory effect was avoided by immunodepletion using 6E10 or A11. In conclusion, our results show evidence of a switch in the activated microglia phenotype from alternative, at the beginning of Aβ pathology, to a classical at advanced stage of the disease in this model. This change was induced, at least in part, by the age-dependent accumulation of extracellular soluble Aβ oligomers. Finally, these cytotoxic activated microglial cells could participate in the neuronal lost observed in AD.

Dopamine D5 receptors of rat and human brain

In contrast to dopamine D1 receptors, the anatomical distribution of D5 receptors in the CNS is poorly described. Therefore, we have studied the localization of dopamine D5 receptors in the brain of rat and human using our newly prepared subtype-specific antibody. Western blot analysis of brain tissues and membranes of cDNA transfected cells, and immunoprecipitation of brain dopamine receptors suggest that this antibody is highly selective for native dopamine D5 receptors. The D5 antibody labeled dopaminergic neurons of mesencephalon, and cortical and subcortical structures. In neostriatum, the D5 receptors were localized in the medium spiny neurons and large cholinergic interneurons. The D5 labeling in caudate nucleus was predominantly in spines of the projection neurons that were frequently making asymmetric synapses. Occasionally, the D5 receptors were also found at the symmetric synapses. Within the cerebral cortex and hippocampus, D5 antibody labeling was prominent in the pyramidal cells and their dendrites. Dopamine D5 receptors were also prominent in the cerebellum, where dopamine innervation is known to be very modest. Differences in the localization of D5 receptors between both species were generally indistinguishable except in hippocampus. In rat, the hippocampal D5 receptor was concentrated in the cell body, whereas in human it was also associated with dendrites. These results show that D5 receptors are localized in the substantia nigra-pars compacta, hypothalamus, striatum, cerebral cortex, nucleus accumbens and olfactory tubercle. Furthermore, the presence of D5 receptors in the areas of dopamine pathways suggests that this receptor may participate actively in dopaminergic neurotransmission.

Early neuropathology of somatostatin/NPY GABAergic cells in the hippocampus of a PS1 × APP transgenic model of Alzheimer's disease

At advanced stages, Alzheimers disease (AD) is characterized by an extensive neuronal loss. However, the early neurodegenerative deficiencies have not been yet identified. Here we report an extensive, selective and early neurodegeneration of the dendritic inhibitory interneurons (oriens-lacunosum moleculare, O-LM, and hilar perforant path-associated, HIPP, cells) in the hippocampus of a transgenic PS1xAPP AD model. At 6 months of age, from 22 different pre- and postsynaptic mRNA markers tested (including GABAergic, glutamatergic and cholinergic markers), only the expression of somatostatin (SOM) and NPY neuropeptides (O-LM and HIPP markers) displayed a significant decrease. Stereological cell counting demonstrated a profound diminution (50-60%) of SOM-immunopositive neurons, preceding the pyramidal cell loss in this AD model. SOM population co-expressing NPY was the most damaged cell subset. Furthermore, a linear correlation between SOM and/or NPY deficiency and Abeta content was also observed. Though the molecular mechanism of SOM neuronal loss remains to be determined, these findings might represent an early hippocampal neuropathology. Therefore, SOM and NPY neuropeptides could constitute important biomarkers to assess the efficacy of potential early AD treatments.

Age-dependent Accumulation of Soluble Amyloid β (Aβ) Oligomers Reverses the Neuroprotective Effect of Soluble Amyloid Precursor Protein-α (sAPPα) by Modulating Phosphatidylinositol 3-Kinase (PI3K)/Akt-GSK-3β Pathway in Alzheimer Mouse Model

Neurotrophins, activating the PI3K/Akt signaling pathway, control neuronal survival and plasticity. Alterations in NGF, BDNF, IGF-1, or insulin signaling are implicated in the pathogenesis of Alzheimer disease. We have previously characterized a bigenic PS1×APP transgenic mouse displaying early hippocampal Aβ deposition (3 to 4 months) but late (17 to 18 months) neurodegeneration of pyramidal cells, paralleled to the accumulation of soluble Aβ oligomers. We hypothesized that PI3K/Akt/GSK-3β signaling pathway could be involved in this apparent age-dependent neuroprotective/neurodegenerative status. In fact, our data demonstrated that, as compared with age-matched nontransgenic controls, the Ser-9 phosphorylation of GSK-3β was increased in the 6-month PS1×APP hippocampus, whereas in aged PS1×APP animals (18 months), GSK-3β phosphorylation levels displayed a marked decrease. Using N2a and primary neuronal cell cultures, we demonstrated that soluble amyloid precursor protein-α (sAPPα), the predominant APP-derived fragment in young PS1×APP mice, acting through IGF-1 and/or insulin receptors, activated the PI3K/Akt pathway, phosphorylated the GSK-3β activity, and in consequence, exerted a neuroprotective action. On the contrary, several oligomeric Aβ forms, present in the soluble fractions of aged PS1×APP mice, inhibited the induced phosphorylation of Akt/GSK-3β and decreased the neuronal survival. Furthermore, synthetic Aβ oligomers blocked the effect mediated by different neurotrophins (NGF, BDNF, insulin, and IGF-1) and sAPPα, displaying high selectivity for NGF. In conclusion, the age-dependent appearance of APP-derived soluble factors modulated the PI3K/Akt/GSK-3β signaling pathway through the major neurotrophin receptors. sAPPα stimulated and Aβ oligomers blocked the prosurvival signaling. Our data might provide insights into the selective vulnerability of specific neuronal groups in Alzheimer disease.

The Subunit Composition of a GABAA/Benzodiazepine Receptor from Rat Cerebellum

Abstract: The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABAA receptors that show diazepam (or clonazepam)‐insensitive [3H]Ro 15‐4513 binding has been determined by immunoprecipitation with subunit‐specific antibodies. These receptors have α6, α1, γ2S, γ2L, and β2 or β3 subunits colocalizing in the same receptor complex.

Abnormal accumulation of autophagic vesicles correlates with axonal and synaptic pathology in young Alzheimer’s mice hippocampus

Dystrophic neurites associated with amyloid plaques precede neuronal death and manifest early in Alzheimer’s disease (AD). In this work we have characterized the plaque-associated neuritic pathology in the hippocampus of young (4- to 6-month-old) PS1M146L/APP751SL mice model, as the initial degenerative process underlying functional disturbance prior to neuronal loss. Neuritic plaques accounted for almost all fibrillar deposits and an axonal origin of the dystrophies was demonstrated. The early induction of autophagy pathology was evidenced by increased protein levels of the autophagosome marker LC3 that was localized in the axonal dystrophies, and by electron microscopic identification of numerous autophagic vesicles filling and causing the axonal swellings. Early neuritic cytoskeletal defects determined by the presence of phosphorylated tau (AT8-positive) and actin–cofilin rods along with decreased levels of kinesin-1 and dynein motor proteins could be responsible for this extensive vesicle accumulation within dystrophic neurites. Although microsomal Aβ oligomers were identified, the presence of A11-immunopositive Aβ plaques also suggested a direct role of plaque-associated Aβ oligomers in defective axonal transport and disease progression. Most importantly, presynaptic terminals morphologically disrupted by abnormal autophagic vesicle buildup were identified ultrastructurally and further supported by synaptosome isolation. Finally, these early abnormalities in axonal and presynaptic structures might represent the morphological substrate of hippocampal dysfunction preceding synaptic and neuronal loss and could significantly contribute to AD pathology in the preclinical stages.

Chemical composition of lipophilic extractives from Eucalyptus globulus Labill. wood

Summary The composition of lipophilic extractives in the chloroform soluble fraction of the acetone extract from Eucalyptus globulus wood has been examined. The lipid extract was fractionated by solid-phase extraction on aminopropyl-phase cartridges into four different fractions of increasing polarity. The total lipid extract and the resulting fractions were analyzed by gas chromatography and gas chromatography-mass spectrometry, using high temperature capillary columns. The main compounds identified included sterols, sterol esters, fatty acids, steroid ketones, hydrocarbons and triglycerides. Minor compounds such as fatty alcohols, mono- and diglycerides, waxes and tocopherols were also identified among the lipids from E. globulus wood.

The α1 and α6 Subunits Can Coexist in the Same Cerebellar GABAA Receptor Maintaining Their Individual Benzodiazepine-Binding Specificities

Abstract: Two GABAA receptor subunit‐specific antibodies anti‐α6 and anti‐α1 have been used for elucidating the relationship between the presence of α1 and/or α6 subunits in the cerebellar GABAA receptors and the benzodiazepine‐binding specificity. Receptor immunoprecipitation with the subunit‐specific antibodies shows that 39% of the cerebellar GABAA receptors have α6, whereas 76% of the receptors have α1 as determined by [3H]muscimol binding. Results show that 42–45% of the receptors having α6 also have α1, whereas 13–15% of the receptors that contain α1 also have α6. The immunoprecipitation results as well as immunopurification and immunoblotting experiments reveal the existence of three types of cerebellar GABAA receptors; i.e., one has both α1 and α6 subunits, a second type has α1 but not α6, and a third type has α6 but not α1 subunits. The results also show that receptors where α1 and α6 subunits coexist have two pharmacologically different benzodiazepine‐binding properties, each associated with a different α subunit. The α1 subunit contributes the high‐affinity binding of [3H]Ro 15‐1788 (flumazenil) and the diazepam‐sensitive binding of [3H]Ro 15‐4513. The α6 subunit contributes the diazepam‐insensitive binding of [3H]Ro 15‐4513, but it does not bind [3H]Ro 15‐1788 with high affinity. Thus, in the cerebellar α1–α6 GABAA receptors, there is no dominance of the pharmacology of one α subunit over the other.

Rat hippocampal GABAergic molecular markers are differentially affected by ageing

We previously reported that the pharmacological properties of the hippocampal GABAA receptor and the expression of several subunits are modified during normal ageing. However, correlation between these post‐synaptic modifications and pre‐synaptic deficits were not determined. To address this issue, we have analysed the mRNA levels of several GABAergic molecular markers in young and old rat hippocampus, including glutamic acid decarboxylase enzymes, parvalbumin, calretinin, somatostatin, neuropeptide Y and vasoactive intestinal peptide (VIP). There was a differential age‐related decrease in these interneuronal mRNAs that was inversely correlated with up‐regulation of the α1 GABA receptor subunit. Somatostatin and neuropeptide Y mRNAs were most frequently affected (75% of the animals), then calretinin and VIP mRNAs (50% of the animals), and parvalbumin mRNA (25% of the animals) in the aged hippocampus. This selective vulnerability was well correlated at the protein/cellular level as analysed by immunocytochemistry. Somatostatin interneurones, which mostly innervate principal cell distal dendrites, were more vulnerable than calretinin interneurones, which target other interneurones. Parvalbumin interneurones, which mostly innervate perisomatic domains of principal cells, were preserved. This age‐dependent differential reduction of specific hippocampal inteneuronal subpopulations might produce functional alterations in the GABAergic tone which might be compensated, at the post‐synaptic level, by up‐regulation of the expression of the α1 GABAA receptor subunit.

Short and Long Form γ2 Subunits of the GABAA/Benzodiazepine Receptors

Abstract: Three novel antisera to the γ2 subunit of the γ‐aminobutyric acidA (GABAA) receptor/benzodiazepine receptor (GABAAR/BZDR) complex have been made. Anti‐γ2S and anti‐γ2L are specific antibodies to synthetic peptides that recognize the γ2S (short) and γ2L (long) forms, respectively, of the γ2 subunit. An antibody (anti‐γ2IL2) to staphylococcal protein A fusion protein of the large intracellular loop (γ2IL) located between the putative transmembrane segments M3 and M4 of γ2S recognizes both γ2S and γ2L subunits. The antibodies immunoprecipitated both the solubilized and affinity‐purified GABAAR/BZDR from rat and bovine brain. Immunoblots with membranes from rat brain cerebral cortex as well as with affinity‐purified receptor from bovine cortex show that anti‐γ2S and anti‐γ2L recognize peptides of 45,000 and 47,000 Mr, respectively. Immunoprecipitation experiments indicate that γ2S is more prevalent in hippocampus, whereas γ2L is more abundant in cerebellum. Intermediate values for each form are found in the cerebral cortex. The results suggest that in the rat brain there is a considerable amount of colocalization of γ2S and γ2L in the same receptor complex. In the cerebral cortex, 15% of the BZDRs contain both γ2S and γ2L subunits and 41–48% of the γ2L subunit coexists with γ2S in the same receptor complex. In cerebellum, in 27% of the clonazepam‐sensitive and 39% of the clonazepam‐insensitive BZDRs the γ2S and γ2L coexist in the same receptor complex. The latter are presumably localized in granule cells and also contain α6. In addition, almost all (93%) the clonazepam‐insensitive BZDRs that contain γ2L also contain a γ2S subunit in the same receptor complex. The most likely interpretation of the results is that there is an important population of granule cell receptors that contain α6, γ2S, and γ2L coexisting in the same receptor complex. Nevertheless, 31% of the cerebellar receptors that contain α6 subunit(s) have neither γ2S nor γ2L subunits. There are also species differences with respect to the relative abundance of γ2S and γ2L. These results might be relevant for understanding the molecular mechanisms underlying some of the GABAAR/BZDR‐mediated effects of ethanol intoxication involving cerebellar granule cells.