Juan F. López-Téllez

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.

Role of layer 6 of V2 visual cortex in object-recognition memory.

Modulating Visual Memory Layer 3 of the secondary visual cortical area V2 plays a role in visual information processing. However, in contrast to layer 3, layer 6 of visual cortex is composed of many types of neurons and their response to visual stimuli is more complex. The importance of layer 6 in visual information processing remains an enigma. López-Aranda et al. (p. 87; see the Perspective by Saksida) investigated its role in the rat visual cortex in the processing of both short- and long-term visual memory. Elimination of neurons from layer 6 of area V2 led to complete loss of normal memory. However, overexpression of a protein called RGS-14 in the same area boosted the visual memory capacity in such a way that the animals retained the visual information for many months, rather than the normal 45 minutes. Experiments reveal the localization of short- and long-term visual memory encoding in the rat visual cortex. Cellular responses in the V2 secondary visual cortex to simple as well as complex visual stimuli have been well studied. However, the role of area V2 in visual memory remains unexplored. We found that layer 6 neurons of V2 are crucial for the processing of object-recognition memory (ORM). Using the protein regulator of G protein signaling–14 (RGS-14) as a tool, we found that the expression of this protein into layer 6 neurons of rat-brain area V2 promoted the conversion of a normal short-term ORM that normally lasts for 45 minutes into long-term memory detectable even after many months. Furthermore, elimination of the same-layer neurons by means of injection of a selective cytotoxin resulted in the complete loss of normal as well as protein-mediated enhanced ORM.

Expression of the scaffolding PDZ protein glutaminase-interacting protein in mammalian brain

A human brain cDNA clone coding for a novel PDZ‐domain protein of 124 amino acids was previously isolated in our laboratory. The protein was termed glutaminase‐interacting protein (GIP), because it interacts with the C‐terminal region of the human L‐type glutaminase (LGA). The pattern of expression and functions of GIP in brain are completely unknown, so its significance remains undefined. Here we describe the expression of GIP mRNA and protein in mammalian brain. Northern blot analysis revealed that GIP mRNA was ubiquitous in most regions of human brain but was particularly abundant in spinal cord. The presence of the protein in rat and monkey brain was studied at the regional, cellular, and subcellular level by immunocytochemistry. The protein was found to be present in both neurons and astrocytes, with a cytosolic and mitochondrial subcellular localization. Double immunofluorescence labeling with anti‐GIP and anti‐LGA antibodies using confocal microscopy revealed colocalization of both proteins in astrocyte cell processes and their perivascular end feet. Electron microscopy of rat brain neurons revealed GIP immunoreactivity concentrated also in the nuclear envelope and the plasma membrane. The multiple locations for GIP in mammalian brain are in agreement with known protein interaction partners reported for this PDZ protein. The findings presented here support a role of GIP as an important scaffold in both astrocytes and neurons and point toward astrocytic processes and perivascular end feet as plausible anatomical substrates for interaction with glutaminase.

Segregation of two glutaminase isoforms in islets of Langerhans

Despite the importance of glutamatergic signalling in the co-ordination of hormone secretion, the identity of the enzyme for the production of glutamate in beta-cells is still unresolved. We have found that the endocrine pancreas co-expresses two isoforms of GA (glutaminase), denoted as kidney-type (KGA) and liver-type (LGA), with a complementary cellular pattern of expression. Whereas KGA was mainly present in alpha-cells, LGA was very abundant in beta-cells. This spatial segregation may have important functional implications, facilitating a differential regulation of glutamate production in insulin- and glucagon-secreting cells.

Activation of caspase-3 pathway by expression of sGαi2 protein in BHK cells

Treatment with dopamine and other dopamine D2 receptor agonists has been shown to induce cell death through activation of caspase-3 pathway. However, initial step that leads to the activation of caspase-3 in D2 receptor-mediated apoptotic pathway remains unclear. Recently, it was shown that a spliced variant of Galphai2 protein (sGalphai2) forms intracellular complex with D2 receptors by protein-protein interaction and that D2 drugs treatment causes the liberation of sGalphai2 protein from complex. Now, we show that the unbound form of sGalphai2 protein is able to activate caspase-3 pathway in baby hamster kidney (BHK) cells. Expression of sGalphai2 protein in BHK cells led to the production of active form of caspase-3 and activation of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular regulated kinase 1/2 (ERK1/2). Co-expression of sGalphai2 with either D2 short (D2S) or D2 long (D2L) isoforms of dopamine D2 receptors blocked the activation of caspase-3 pathway. Thus, our results demonstrate that high level of unbound sGalphai2 protein can affect the cell survival and engagement of this protein with D2 receptors can block this process. It is suggested that this process may be a crucial step in the initiation of D2 receptor-mediated cellular apoptosis through this pathway.

Regulator of G-protein signaling 14 protein modulates Ca2+ influx through Cav1 channels

Calcium flux through L-type voltage-activated calcium (Cav1) channels is crucial for regulating brain functions including memory formation and behavior. Alterations in Ca2+ homeostasis have been linked to many cognitive disorders, and understanding the regulation of this process is crucial for their remedy. Therefore, here, we have evaluated the effect of a multifunctional protein known to be involved in memory functions called regulator of G-protein signaling 14 (RGS-14) on Cav1 channel activity in neuronal cell lines NG108-15 and SH-SY5Y. RGS-14 protein produced significant reduction in Ca2+ influx in both cell lines and this effect was dependent on nifedipine-sensitive Cav1 channels. Thus, our results provide evidence supporting the idea that RGS-14 may facilitate the cognitive processing by modulating Cav1 channel-mediated intracellular Ca2+ transients.

A correlation of haloperidol-induced cognitive deficit with dysfunctional dopamine receptor activity in nonhuman primate

Haloperidol is an antipsychotic drug that acts through blockage of dopamine D 2 receptors. Chronic administration of this antipsychotic drug in nonhuman primates induces a pronounced cognitive deficit. However, receptor subtypes that are responsible for this cogni - tive dysfunction remain unknown. Therefore, brains of chronic haloperidol-treated young and aged monkeys were used to analyze the intricate relation of receptor activity, cognitive dysfunction, and haloperidol-mediated actions in the production of harmful effects. Tak- ing into account the significant cognitive loss observed after haloperidol treatment, it was predicted that changes in the cognitive status that correlate with the receptor activity in the prefrontal cortex and striatum, areas implicated in the processing of haloperidol-mediated effects in brain, should be common in both young and aged animals. Based on this concept, we observed that in the prefrontal cortex, dopamine D 1 and D 2 receptors showed changes in receptor levels that were common in both age groups. However, this relationship was absent in GABA A , serotonin 5HT2 and muscaranic receptors. In contrast to the prefrontal cortex, in striatum, this change was restricted to the dopamine D 2 receptors only. Therefore, from our results, it seems that apart from the downregulation of D 1 receptor activity in the prefrontal cortex, an upregulation of D 2 receptors could also contribute to the generation of the cognitive loss observed in haloperidol-treated monkeys. Additionally, reduced excitatory input due to hampered cortico-striatal D 1 dopaminergic activity and stronger inhibition at the synapse of excitatory input site by upregulated striatal D 2 receptor activity could promote the side effects

A dynamic expression pattern of sGαi2 protein during early period of postnatal rat brain development

The function of sGαi2 protein in central nervous system is not well understood. Therefore to explore the possible role of this protein in postnatal brain development, we have analyzed the protein expression pattern of brain obtained from rats of postnatal day 0 (P0) to P90 by dot‐blots and immunocytochemistry techniques. In dot‐blots, both nuclear and membrane fractions showed a gradual decrease from P0 to P60. Highest protein level was observed at the age of P0. There was also a trend of decline in the sGαi2 protein from P0 to P90 in brain sections stained by immunocytochemistry method. At P0, the protein labeling was highest in cerebral cortex, hippocampus, cerebellum and mitral cell layer. In cerebral cortex, a drop in the immunolabeling of sGαi2 protein was observed at P3, which was significantly increased at the age of P5. However, in striatum and olfactory tubercle, it was maintained through P0–P10 and P0–P5, respectively. Thalamus was one of the areas where labeling was not as strong as cortex, hippocampus or striatum. In contrary to other areas, immunostaining of sGαi2 in corpus‐callosum and lacunosum‐moleculare was not seen at P0 and appeared in advanced postnatal ages. A detectable level of sGαi2 protein was observed at P5 in carpus‐callosum and at P20 in lacunosum‐moleculare. A high level of sGαi2 protein in the period when cellular layer organization and synaptic innervations, synaptic connections and maturation take place, suggests for a potential role of this protein in the early postnatal brain development.