Javier Vitorica

Cellular environment facilitates protein accumulation in aged rat hippocampus

Aging represents the main risk factor to develop Alzheimer disease (AD) and protein aggregation constitutes a pathological hallmark thought to be involved in the etiology of this disease. Here, we show that, in basal conditions, the expression of chaperones calnexin, protein disulfide isomerase (PDI) and Grp78 was decreased in aged hippocampus, whereas the protein ubiquitination increased, suggesting the existence of age-related deficits in the systems involved in the defense against unfolded proteins. Interestingly, when cellular stress was induced by intra-hippocampal lactacystin injection, the aged rats were less efficient than young animals in alleviating the protein accumulation and, as an important factor, did not induce the expression of chaperones as young animals. However, the expression of the pro-apoptotic factor CHOP/GADD153 was induced and caspase-12 was activated in stressed aged rats but not in young animals. Current results demonstrated that unfolding protein response (UPR) is not correctly activated in aged rat hippocampus. Consequently, the up-regulation of apoptotic pathway mediators is increased in aged rats. Results might provide further understanding of the pathogenic mechanisms of age-related neurodegenerative disorders.

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.

Comparative autoradiographic distribution of central ω (benzodiazepine) modulatory site subtypes with high, intermediate and low affinity for zolpidem and alpidem

Pharmacological characterization of [3H]benzodiazepine binding to membrane preparations of adult rat hippocampus and neonatal rat brain have demonstrated, in addition to the omega 1 and omega 2 populations of central omega benzodiazepine binding sites associated with GABAA receptors, the existence of binding sites with microM affinity for the imidazopyridines zolpidem and alpidem. In the present study we have investigated their comparative autoradiographic distribution using [3H]flumazenil as a ligand. In the neonatal rat CNS, the imidazopyridine derivatives zolpidem and alpidem were found to discriminate two [3H]flumazenil binding site populations with an IC50 value ratio of more than 200-fold. In the different regions investigated (spinal cord, striatum, neocortex and inferior colliculus) the low affinity component had IC50 values of 20-40 microM (zolpidem) and 5-15 microM (alpidem) and accounted for ca. 50% of the total binding site population. In the adult rat, these imidazopyridine derivatives displayed a greater displacing potency in the cerebellum (IC50 = 6 and 36 nM, respectively) than in the hippocampus (IC50 = 37 and 403 nM, respectively). In the cerebellum, [3H]flumazenil binding was fully displaced by 1 microM of either compound and Hill coefficients of displacement curves were close to unity. In the hippocampus, 25% of [3H]flumazenil binding were resistant to 3 microM zolpidem or 1 microM alpidem, but were displaced by 100 microM of either compound. CL 218,872 also displayed a greater displacing potency in the cerebellum (IC50 = 83 nM) than in the hippocampus (IC50 = 711 nM) but [3H]flumazenil binding in the hippocampus was fully displaced by 10 microM of this compound. In adult rat hippocampus, zolpidem and alpidem were found to discriminate between three central omega site subtypes which display high (IC50 = 31 and 6.1 nM, for these imidazopyridine derivatives. In contrast, CL 218,872 discriminated between omega 1 and omega 2 sites but not between two omega 2 receptor subpopulations. omega 1 sites were mainly localized in layer IV of the sensorimotor cortex, cerebellum, substantia nigra, olfactory bulb and inferior colliculus. omega 2I sites were present in the cortical mantle (with higher levels in the cingulate and olfactory than in the sensorimotor cortex) and in subcortical (hippocampus, hypothalamus and nucleus accumbens) limbic structures. In the hippocampus, hypothalamus, spinal cord and nucleus accumbens, omega 2L sites accounted for more than 25% of the specific [3H]flumazenil binding; the density of these sites was minor in the cortex and in most pyramidal and extrapyramidal system structures.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Differential expression of the short and long forms of the γ2 subunit of the GABAA/benzodiazepine receptors

The distribution of the mRNAs encoding the gamma 2S and gamma 2L subunits of the GABAA receptor in the rat brain has been revealed by in situ hybridization, northern blot and dot blot analysis using specific antisense oligonucleotides. In addition, the quantitative distribution of the gamma 2S and gamma 2L subunit peptides participating in the fully assembled GABAA receptors/benzodiazepine receptors has been mapped by immunoprecipitation with specific anti-gamma 2S and anti-gamma 2L antibodies. Several neuronal types and brain regions are enriched in gamma 2L such as neurons of the layer II of striate cortex and cerebellar Purkinje cells as well as the inferior colliculus, superior colliculus, deep cerebellar nuclei, medulla and pons. Other neuronal types and regions are enriched in gamma 2S such as the mitral cells of the olfactory bulb, pyramidal neurons of the pyriform cortex, layer VI of the neocortex, granule cells of the dentate gyrus and pyramidal cells of the hippocampus. Other cortical areas and cerebellar granule cells express both gamma 2S and gamma 2L in comparable amounts. There is a good correlation between the relative expression of gamma 2S and gamma 2L mRNAs and the relative presence of these protein subunits in fully assembled and mature receptors in the studied brain regions. The differential distribution of gamma 2S and gamma 2L might result in differential ethanol sensitivity of the neurons expressing these GABAA receptor subunits.

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.

Heterogeneity in the Allosteric Interaction Between the γ-Aminobutyric Acid (GABA) Binding Site and Three Different Benzodiazepine Binding Sites of the GABAA/Benzodiazepine Receptor Complex in the Rat Nervous System

Abstract: In the present communication we have investigated the allosteric coupling between the γ‐aminobutyric acidA (GABAA) receptor and the pharmacologically different benzodiazepine (BZD) receptor subtypes in membranes from various rat nervous system regions. Two types of BZD receptors (type I and type II) have been classically defined using CL 218.872. However, using zolpidem, three different BZD receptors have been identified by binding displacement experiments in membranes. These BZD receptor subtypes displayed high, low, and very low affinity for zolpidem. The distribution of the high‐ and low‐affinity binding sites for zolpidem was similar to that of type I and type II subtypes in cerebellum, prefrontal cortex, and adult cerebral cortex. On the other hand, the very‐low‐affinity binding site was localized in relative high proportion in spinal cord, hippocampus, and newborn cerebral cortex and, to a minor extent, in superior colliculus. The allosteric coupling between the GABAA receptor and the BZD receptor subtypes was different. The high‐ and low‐affinity binding sites for zolpidem seemed to have a similar high degree of coupling, except in spinal cord. On the other hand, the very‐low‐affinity binding site for zolpidem displayed a low degree of coupling with the GABAA receptor. These results seem to indicate that the different efficacy of GABA in enhancing the [3H]flunitrazepam binding could be due to the different BZD receptor subtypes present in the GABAA/BZD receptor complex and, moreover, led us to speculate that the low GABA efficacy found in membranes from spinal cord, hippocampus, and newborn cerebral cortex might be due to the presence in relatively high proportion of the very‐low‐affinity binding site for zolpidem.

Cytosolic free calcium levels increase with age in rat brain synaptosomes.

Calcium homeostasis in synaptosomes is altered during ageing. The cytosolic free calcium concentration, [Ca2+]i was determined in synaptosomes and crude synaptosomal fractions from 3- and 24-month-old rats with the fluorescent indicator quin-2. The [Ca2+]i were around two times higher in 24-month-old rats than in adults, both under resting conditions and after K depolarization. This difference was still observed after incubation with an endogenous heavy metal chelator. To avoid the calcium buffering effect of quin-2, [Ca2+]i values were determined with the use of a null-point method and with fura-2. These methods confirmed the increase in [Ca2+]i with age in synaptosomes. The increase in [Ca2+]i in nerve endings may be pathologically important in brain ageing.

Immunohistochemical localization of GABAA receptors in the retina of the new world primate Saimiri sciureus.

A large population of amacrine cells in the retina are thought to use GABA as an inhibitory neurotransmitter in their synaptic interactions within the inner plexiform layer. However, little is known about their synaptic targets; the neurons that express the receptors for GABA have not been clearly identified. Recently, the GABAA receptor has been isolated and antibodies have been raised against it. These antibodies have proven useful for the immunocytochemical localization of the receptor, and two brief reports describing the distribution of GABAA receptor immunoreactivity in the retina have appeared (Richards et al., 1987; Mariani et al., 1987). We used a monoclonal antibody (62-3G1) against the GABAA receptor to study the retina of the New World primate Saimiri sciureus. Labeled somata were found in the inner nuclear layer (INL) and ganglion cell layer (GCL). The staining was confined to what appeared to be the cells plasmalemma and small cytoplasmic granules. Most of the labeled neurons in the INL had small somata (5-7 microns in diameter) located at the vitreal edge of the layer. They arborized in two laminae (approximately 2 and 4) of inner plexiform layer (IPL). Ventral to the optic disc (2.5 mm) they comprised 29% of the cells present. A few of the labeled neurons appeared to be interplexiform cells or flat bipolar cells, with labeled processes that extended into both the IPL and the inner half of the outer plexiform layer. In the GCL, the labeled somata were among the largest present (13-20 microns in diameter), and 2.5 mm ventral to the optic disc they made up 15% of the cells present. Experiments in which immunoreactive somata were retrogradely labeled following the injection of fluorescent tracers into the optic tract provided a conclusive demonstration that some of the immunoreactive somata were ganglion cells. The antibody often labeled their axons in the optic fiber layer. This suggests that the GABAA receptors are transported anterogradely to the retinal terminal fields. The dendrites of the immunoreactive ganglion cells extended into the 2 laminae of labeled processes in the IPL, and their primary dendritic arbors were, at any given eccentricity, quite similar in appearance. This homogeneity suggests that they comprise a particular subset of the ganglion cells. Sections simultaneously labeled with the monoclonal antibody against the GABAA receptor and antisera against either L-glutamic acid decarboxylase (GAD) or GABA revealed that the GAD/GABA was distributed much more widely in the IPL than the GABAA receptor.(ABSTRACT TRUNCATED AT 400 WORDS)