Retrograde and anterograde contextual fear amnesia induced by selective elimination of layer IV-Va neurons in the granular retrosplenial cortex (A29).

Differences in cytoarchitectural organization and connectivity distinguishes granular (or area 29, A29) and dysgranular (or area 30, A30) subdivisions of the retrosplenial cortex (RSC). Although increasing evidence supports the participation of RSC in contextual fear learning and memory, the contribution of each RSC subdivision remains unknown. Here we used orchiectomized rats and intraperitoneal (i.p.) injections of saline (control) or 5 mg/kg MK801, to trigger selective degeneration of pyramidal neurons in layers IV-Va of A29 (A29MK801 neurons). These treatments were applied 3 days before or two days after contextual fear conditioning, and contextual fear memory was evaluated by scoring freezing in the conditioned context five days after training. Afterwards, brains were fixed and c-Fos and Egr-1 expression were assessed as surrogates of neuronal activity elicited by the recall in A29, A30 and in limbic areas. We found that eliminating A29MK801 neurons after training reduces conditioned freezing to 43.1 ± 9.9% respect to control rats. This was associated with a significant reduction of c-Fos and Egr-1 expression in A30, but not in other limbic areas. On the other hand, eliminating A29MK801 neurons before training caused a mild but significant reduction of conditioned freezing to 79.7 ± 6.8%, which was associated to enhanced expression of c-Fos in A29, A30 and CA1 field of hippocampus, while Egr-1 expression in caudomedial (CEnt) entorhinal cortex was not depressed as in control animals. These observations show that severeness of amnesia differs according to whether A29MK801 neurons were eliminated before or after conditioning, likely because loss of A29MK801 neurons after conditioning disrupt memory engram while their elimination before training allow recruitment of other neurons in A29 for partial compensation of contextual fear learning and memory. These observations add further support for the critical role of A29MK801 neurons in contextual fear learning and memory by connecting limbic structures with A30.

Fear-context association during memory retrieval requires input from granular to dysgranular retrosplenial cortex.

The contribution of the granular (area 29, A29) and dysgranular (area 30, A30) subdivisions of the retrosplenial cortex (RSC) to contextual fear memory (CFM) retrieval remains elusive. Here, intact and orchiectomized (ORC) male rats received an intraperitoneal (I.P.) injection of saline (control) or 5 mg/Kg MK801 after training and memory formation. In ORC, but not in intact males, this MK801 treatment selectively induces overt loss of neurons in layers IV-Va of A29 (A29MK801 neurons) (Sigwald et al., 2016). Compared to ORC-saline, ORC-MK801 rats showed impaired CFM retrieval in an A-B-A design for contextual fear conditioning (CFC), however context recognition was not affected. In ORC-MK801 rats, neither novel object recognition nor object-in-context discrimination were impaired, further indicating that A29MK801 neurons are not required for contextual recognition. Elevated plus maze test showed that anxiety-like behavior was not affected in ORC-MK801 animals, suggesting that loss of A29MK801 neurons does not affect the emotional state that could impair freezing during test. Importantly, in a sensory preconditioning test, higher order CFM retrieval was abolished in ORC-MK801, but not in male-MK801. Collectively, these observations indicate that A29MK801 neurons are critically required for retrieving fear-context association. For dissecting the anatomofunctional contribution of A29MK801 neurons to CFM retrieval, expression of c-Fos and Egr-1 was used to map brain-wide neuronal activity. In control male rats CFC and CFM retrieval was associated with significant enhancement of both proteins in limbic structures and A30, but not in A29, suggesting that neurons in A30 and limbic structures encode and store the associative experience. Notably, in ORC but not in intact males, MK801 impairs CFM retrieval and expression of c-Fos and Egr-1 proteins in A30, without affecting their expression in limbic structures. Thus, the loss of A29MK801 neurons after CFM formation precludes activation of associative neurons in A30, impairing CFM recall. FluoroGold retrograde track-tracing confirmed that A29MK801 neurons project to A30. Silver staining provide evidence that MK801 in ORC rats induces axonal deafferentation of A29MK801 neuron in A30. Collectively, our experiments provide the first evidence that A30 neurons participate in encoding and storing CFM while A29 is required for their activation during recall.

Wnt-5a/Frizzled9 Receptor Signaling through the Gαo-Gßγ Complex Regulates Dendritic Spine Formation.

Wnt ligands play crucial roles in the development and regulation of synapse structure and function. Specifically, Wnt-5a acts as a secreted growth factor that regulates dendritic spine formation in rodent hippocampal neurons, resulting in postsynaptic development that promotes the clustering of the PSD-95 (postsynaptic density protein 95). Here, we focused on the early events occurring after the interaction between Wnt-5a and its Frizzled receptor at the neuronal cell surface. Additionally, we studied the role of heterotrimeric G proteins in Wnt-5a-dependent synaptic development. We report that FZD9 (Frizzled9), a Wnt receptor related to Williams syndrome, is localized in the postsynaptic region, where it interacts with Wnt-5a. Functionally, FZD9 is required for the Wnt-5a-mediated increase in dendritic spine density. FZD9 forms a precoupled complex with Gαo under basal conditions that dissociates after Wnt-5a stimulation. Accordingly, we found that G protein inhibition abrogates the Wnt-5a-dependent pathway in hippocampal neurons. In particular, the activation of Gαo appears to be a key factor controlling the Wnt-5a-induced dendritic spine density. In addition, we found that Gßγ is required for the Wnt-5a-mediated increase in cytosolic calcium levels and spinogenesis. Our findings reveal that FZD9 and heterotrimeric G proteins regulate Wnt-5a signaling and dendritic spines in cultured hippocampal neurons.

The physiological role of the amyloid precursor protein as an adhesion molecule in the developing nervous system.

The amyloid precursor protein (APP) is a type I transmembrane glycoprotein better known for its participation in the physiopathology of Alzheimer disease as the source of the beta amyloid fragment. However, the physiological functions of the full length protein and its proteolytic fragments have remained elusive. APP was first described as a cell-surface receptor; nevertheless, increasing evidence highlighted APP as a cell adhesion molecule. In this review, we will focus on the current knowledge of the physiological role of APP as a cell adhesion molecule and its involvement in key events of neuronal development, such as migration, neurite outgrowth, growth cone pathfinding, and synaptogenesis. Finally, since APP is over-expressed in Down syndrome individuals because of the extra copy of chromosome 21, in the last section of the review, we discuss the potential contribution of APP to the neuronal and synaptic defects described in this genetic condition. Read the Editorial Highlight for this article on page 9. Cover Image for this issue: doi. 10.1111/jnc.13817.

Developing the theory of the extended amygdala with the use of the cupric-silver technique.

The amygdaloid complex is a crucial component of the basal forebrain that participates in the modulation of many homeostatic functions, emotional behaviors, and learning. These features require a widespread pattern of connections with several brain structures. In the past, the amygdaloid complex was divided into corticomedial and basolateral groups. The existence of a neuronal continuum linking the central amygdaloid nucleus to the lateral bed nucleus of stria terminalis through the subpallidal area was first revealed by José de Olmos (1932-2008) with the aid of his cupric-silver technique. This observation gave birth to the concept of the extended amygdala, a conceptual framework that is useful for understanding the anatomofunctional organization of the amygdaloid complex, with relevance for basic neuroscience and clinical interventions. Traditional tract-tracing staining methods were complicated and tedious to reproduce. Axonal terminal endings were lost among a myriad of normal fibers. The need to visualize these terminals drove de Olmos to develop cupric-silver methods that revealed disintegrating synaptic terminals, without staining normal fibers. In this article, we describe the historical events leading to the development of the cupric-silver technique that evolved into the amino-cupric-silver technique, which developed hand-in-hand over the years.

Axonal transport of APP and the spatial regulation of APP cleavage and function in neuronal cells.

Over two decades have passed since the original discovery of amyloid precursor protein (APP). While physiological function(s) of APP still remain a matter of debate, consensus exists that the proteolytic processing of this protein represents a critical event in the life of neurons and that abnormalities in this process are instrumental in Alzheimer's disease (AD) pathogenesis. Specific molecular components involved in APP proteolysis have been identified, and their enzymatic activities characterized in great detail. As specific proteolytic fragments of APP are identified and novel physiological effects for these fragments are revealed, more obvious becomes our need to understand the spatial organization of APP proteolysis. Valuable insights on this process have been obtained through the study of non-neuronal cells. However, much less is known about the topology of APP processing in neuronal cells, which are characterized by their remarkably complex cellular architecture and extreme degree of polarization. In this review, we discuss published literature addressing various molecular mechanisms and components involved in the trafficking and subcellular distribution of APP and APP secretases in neurons. These include the relevant machinery involved in their sorting, the identity of membranous organelles in which APP is transported, and the molecular motor-based mechanisms involved in their translocation. We also review experimental evidence specifically addressing the processing of APP at the axonal compartment. Understanding neuron-specific mechanisms of APP processing would help illuminating the physiological roles of APP-derived proteolytic fragments and provide novel insights on AD pathogenesis.

Aß Assemblies Promote Amyloidogenic Processing of APP and Intracellular Accumulation of Aß42 Through Go/Gßγ Signaling.

Alzheimer's disease (AD) is characterized by the deposition of aggregated species of amyloid beta (Aß) in the brain, which leads to progressive cognitive deficits and dementia. Aß is generated by the successive cleavage of the amyloid precursor protein (APP), first by ß-site APP cleaving enzyme 1 (BACE1) and subsequently by the γ-secretase complex. Those conditions which enhace or reduce its clearance predispose to Aß aggregation and the development of AD. In vitro studies have demonstrated that Aß assemblies spark a feed-forward loop heightening Aß production. However, the underlying mechanism remains unknown. Here, we show that oligomers and fibrils of Aß enhance colocalization and physical interaction of APP and BACE1 in recycling endosomes of human neurons derived from induced pluripotent stem cells and other cell types, which leads to exacerbated amyloidogenic processing of APP and intracellular accumulation of Aß42. In cells that are overexpressing the mutant forms of APP which are unable to bind Aß or to activate Go protein, we have found that treatment with aggregated Aß fails to increase colocalization of APP with BACE1 indicating that Aß-APP/Go signaling is involved in this process. Moreover, inhibition of Gßγ subunit signaling with ßARKct or gallein prevents Aß-dependent interaction of APP and BACE1 in endosomes, ß-processing of APP, and intracellular accumulation of Aß42. Collectively, our findings uncover a signaling mechanism leading to a feed-forward loop of amyloidogenesis that might contribute to Aß pathology in the early stages of AD and suggest that gallein could have therapeutic potential.

Secreted amyloid precursor protein and holo-APP bind amyloid beta through distinct domains eliciting different toxic responses on hippocampal neurons.

Amyloid beta (Abeta) is a metabolic product of Abeta precursor protein (APP). Deposition of Abeta in the brain and neuronal degeneration are characteristic hallmarks of Alzheimer's disease (AD). Abeta induces neuronal degeneration, but the mechanism of neurotoxicity remains elusive. Increasing evidence implicates APP as a receptor-like protein for Abeta fibrils (fAbeta). In this study, we present further experimental support for the direct interaction of APP with fAbeta and for its involvement in Abeta neurotoxicity. Using recombinant purified holo-APP (h-APP), we have shown that it directly binds fAbeta. Employing deletion mutant forms of APP, we show that two different sequences are involved in the binding of APP to fAbeta. One sequence in the n-terminus of APP is required for binding of fAbeta to secreted APP (s-APP) but not to h-APP. In addition, the extracellular juxtamembrane Abeta-sequence mediates binding of fAbeta to h-APP but not to s-APP. Deletion of the extracellular juxtamembrane Abeta sequence abolishes abnormal h-APP accumulation and toxicity induced by fAbeta deposition, whereas deletions in the n-terminus of APP do not affect Abeta toxicity. These experiments show that interaction of toxic Abeta species with its membrane-anchored parental protein promotes toxicity in hippocampal neurons, adding further support to an Abeta-receptor-like function of APP directly implicated in neuronal degeneration in AD.

Nrf2 stabilization prevents critical oxidative damage in Down syndrome cells.

Mounting evidence implicates chronic oxidative stress as a critical driver of the aging process. Down syndrome (DS) is characterized by a complex phenotype, including early senescence. DS cells display increased levels of reactive oxygen species (ROS) and mitochondrial structural and metabolic dysfunction, which are counterbalanced by sustained Nrf2-mediated transcription of cellular antioxidant response elements (ARE). Here, we show that caspase 3/PKCδdependent activation of the Nrf2 pathway in DS and Dp16 (a mouse model of DS) cells is necessary to protect against chronic oxidative damage and to preserve cellular functionality. Mitochondria-targeted catalase (mCAT) significantly reduced oxidative stress, restored mitochondrial structure and function, normalized replicative and wound healing capacity, and rendered the Nrf2-mediated antioxidant response dispensable. These results highlight the critical role of Nrf2/ARE in the maintenance of DS cell homeostasis and validate mitochondrial-specific interventions as a key aspect of antioxidant and antiaging therapies.

APP/Go protein Gßγ-complex signaling mediates Aß degeneration and cognitive impairment in Alzheimer's disease models.

Deposition of amyloid-ß (Aß), the proteolytic product of the amyloid precursor protein (APP), might cause neurodegeneration and cognitive decline in Alzheimer's disease (AD). However, the direct involvement of APP in the mechanism of Aß-induced degeneration in AD remains on debate. Here, we analyzed the interaction of APP with heterotrimeric Go protein in primary hippocampal cultures and found that Aß deposition dramatically enhanced APP-Go protein interaction in dystrophic neurites. APP overexpression rendered neurons vulnerable to Aß toxicity by a mechanism that required Go-Gßγ complex signaling and p38-mitogen-activated protein kinase activation. Gallein, a selective pharmacological inhibitor of Gßγ complex, inhibited Aß-induced dendritic and axonal dystrophy, abnormal tau phosphorylation, synaptic loss, and neuronal cell death in hippocampal neurons expressing endogenous protein levels. In the 3xTg-AD mice, intrahippocampal application of gallein reversed memory impairment associated with early Aß pathology. Our data provide further evidence for the involvement of APP/Go protein in Aß-induced degeneration and reveal that Gßγ complex is a signaling target potentially relevant for developing therapies for halting Aß degeneration in AD.

Phosphorylation of actin-depolymerizing factor/cofilin by LIM-kinase mediates amyloid beta-induced degeneration: a potential mechanism of neuronal dystrophy in Alzheimer's disease.

Deposition of fibrillar amyloid beta (fAbeta) plays a critical role in Alzheimer's disease (AD). We have shown recently that fAbeta-induced dystrophy requires the activation of focal adhesion proteins and the formation of aberrant focal adhesion structures, suggesting the activation of a mechanism of maladaptative plasticity in AD. Focal adhesions are actin-based structures that provide a structural link between the extracellular matrix and the cytoskeleton. To gain additional insight in the molecular mechanism of neuronal degeneration in AD, here we explored the involvement of LIM kinase 1 (LIMK1), actin-depolymerizing factor (ADF), and cofilin in Abeta-induced dystrophy. ADF/cofilin are actin-binding proteins that play a central role in actin filament dynamics, and LIMK1 is the kinase that phosphorylates and thereby inhibits ADF/cofilin. Our data indicate that treatment of hippocampal neurons with fAbeta increases the level of Ser3-phosphorylated ADF/cofilin and Thr508-phosphorylated LIMK1 (P-LIMK1), accompanied by a dramatic remodeling of actin filaments, neuritic dystrophy, and neuronal cell death. A synthetic peptide, S3 peptide, which acts as a specific competitor for ADF/cofilin phosphorylation by LIMK1, inhibited fAbeta-induced ADF/cofilin phosphorylation, preventing actin filament remodeling and neuronal degeneration, indicating the involvement of LIMK1 in Abeta-induced neuronal degeneration in vitro. Immunofluorescence analysis of AD brain showed a significant increase in the number of P-LIMK1-positive neurons in areas affected with AD pathology. P-LIMK1-positive neurons also showed early signs of AD pathology, such as intracellular Abeta and pretangle phosphorylated tau. Thus, LIMK1 activation may play a key role in AD pathology.

Prion protein inhibits fast axonal transport through a mechanism involving casein kinase 2.

Prion diseases include a number of progressive neuropathies involving conformational changes in cellular prion protein (PrPc) that may be fatal sporadic, familial or infectious. Pathological evidence indicated that neurons affected in prion diseases follow a dying-back pattern of degeneration. However, specific cellular processes affected by PrPc that explain such a pattern have not yet been identified. Results from cell biological and pharmacological experiments in isolated squid axoplasm and primary cultured neurons reveal inhibition of fast axonal transport (FAT) as a novel toxic effect elicited by PrPc. Pharmacological, biochemical and cell biological experiments further indicate this toxic effect involves casein kinase 2 (CK2) activation, providing a molecular basis for the toxic effect of PrPc on FAT. CK2 was found to phosphorylate and inhibit light chain subunits of the major motor protein conventional kinesin. Collectively, these findings suggest CK2 as a novel therapeutic target to prevent the gradual loss of neuronal connectivity that characterizes prion diseases.

Selective neuronal degeneration in the retrosplenial cortex impairs the recall of contextual fear memory.

The retrosplenial cortex (RSC) is one of the largest cortical areas in rodents, and is subdivided in two main regions, A29 and A30, according to their cytoarchitectural organization and connectivities. However, very little is known about the functional activity of each RSC subdivision during the execution of complex cognitive tasks. Here, we used a well-established fear learning protocol that induced long-lasting contextual fear memory and showed that during evocation of the fear memory, the expression of early growth response gene 1 was up-regulated in A30, and in other brain areas implicated in fear and spatial memory, however, was down-regulated in A29, including layers IV and V. To search for the participation of A29 on fear memory, we triggered selective degeneration of neurons within cortical layers IV and V of A29 by using a non-invasive protocol that takes advantage of the vulnerability that these neurons have MK801-toxicity and the modulation of this neurodegeneration by testosterone. Application of 5 mg/kg MK801 in intact males induced negligible neuronal degeneration of A29 neurons and had no impact on fear memory retrieval. However, in orchiectomized rats, 5 mg/kg MK801 induced overt degeneration of layers IV-V neurons of A29, significantly impairing fear memory recall. Degeneration of A29 neurons did not affect exploratory or anxiety-related behavior nor altered unconditioned freezing. Importantly, protecting A29 neurons from MK801-toxicity by testosterone preserved fear memory recall in orchiectomized rats. Thus, neurons within cortical layers IV-V of A29 are critically required for efficient retrieval of contextual fear memory.

Amyloid ß precursor protein as a molecular target for amyloid ß--induced neuronal degeneration in Alzheimer's disease.

A role of amyloid ß (Aß) peptide aggregation and deposition in Alzheimer's disease (AD) pathogenesis is widely accepted. Significantly, abnormalities induced by aggregated Aß have been linked to synaptic and neuritic degeneration, consistent with the "dying-back" pattern of degeneration that characterizes neurons affected in AD. However, molecular mechanisms underlying the toxic effect of aggregated Aß remain elusive. In the last 2 decades, a variety of aggregated Aß species have been identified and their toxic properties demonstrated in diverse experimental systems. Concurrently, specific Aß assemblies have been shown to interact and misregulate a growing number of molecular effectors with diverse physiological functions. Such pleiotropic effects of aggregated Aß posit a mayor challenge for the identification of the most cardinal Aß effectors relevant to AD pathology. In this review, we discuss recent experimental evidence implicating amyloid ß precursor protein (APP) as a molecular target for toxic Aß assemblies. Based on a significant body of pathologic observations and experimental evidence, we propose a novel pathologic feed-forward mechanism linking Aß aggregation to abnormalities in APP processing and function, which in turn would trigger the progressive loss of neuronal connectivity observed early in AD.

Comparative analyses of the neurodegeneration induced by the non-competitive NMDA-receptor-antagonist drug MK801 in mice and rats.

Non-competitive NMDA-receptor-antagonist drugs such as dizocilpine (MK801) induce behavioral changes and neurotoxicity that have made an impact in different fields of neuroscience. New approaches in research use transgenic mice to elucidate cellular mechanisms and circuits involved in the effects of these drugs. However, the neurodegeneration induced by these drugs has been extensively studied in rats, but the data in mice is limited. Therefore it is important to characterize if the neurotoxic pattern in mice corresponds to that of rats. A comparative analysis of the neurodegeneration induced by MK801 (10mg/kg) between Wistar rats, and CD-1, CF-1, and C57BL/6-129/Sv mice of both sexes, at different survival times (15, 24, 32, 48, 56 and 72 h) was analysed with the amino-cupric-silver and fluoro-jade B techniques. To compare different administration patterns, groups of mice received subchronic treatments with different doses (final doses of 20 and 40 mg/kg). Results showed that mice treated with MK801 presented different neurotoxic profiles, such as excitotoxic-like cell death in the retrosplenial cortex, terminal degeneration in CA1 and apoptotic-like degeneration in the olfactory bulb. Unlike rats, mice subjected to the same treatment failed to show neurodegeneration in corticolimbic areas such as piriform cortex and dentate gyrus. The amount of degeneration was lower in mice, and the subchronic administration of MK801 did not change the neurotoxic pattern. Additionally, mice lacked the sexually dimorphic response to MK801 toxicity observed in rats. Altogether these results indicate important species dissimilarities. Neurotoxicological studies aimed to explore pathways and mechanisms of MK801 toxicity should consider these differences when using mice as rodent models.

Momento quirúrgico apoyado con doppler transcraneal en pacientes con hemorragia subaracnoidea aneurismática / Surgical timing supported by transcranial doppler in patients with subarachnoid hemorrhage from aneurisms rupture

Introducción: La cirugía de la hemorragia subaracnoidea aneurismática (HSA) realizada en las primeras 72 horas es beneficiosa. Cuando los casos arriban transcurrido este período el mejor momento quirúrgico es controversial. Objetivo. Evaluar la influencia sobre los resultados de la cirugía en la HSA de un protocolo para decidir el momento quirúrgico apoyado en el monitoreo con Doppler transcraneal (DTC). Material y Método: Se comparan los resultados quirúrgicos al alta y al año de seguimiento según la escala de Glasgow para resultados (EGR), en una serie de 233 casos con HSA rotos operados Enero de 2006 - Diciembre de 2010 y seguidos hasta Enero de 2012, en los que la cirugía en el período intermedio se decidió teniendo en cuenta las velocidades de flujo de los segmentos proximales del polígono de Willis registradas por DTC, con los de un grupo control histórico operado Diciembre de 1983 - Diciembre de 2005 sin la ayuda de dicho monitoreo. Resultados: La mortalidad al alta y al año en la serie de estudio fue de 4,3 y 4,5 por ciento y en el grupo control 7 y 7,7 por ciento respectivamente. Se observaron resultados satisfactorios (grados 4 y 5 en EGR) en el 93,1 al alta y 92,8 por ciento al año en la serie de estudio. Entre los controles históricos estos índices fueron 85,6 y 88,1 por ciento respectivamente (p = 0,004 y p = 0,036). Conclusiones: Los resultados del tratamiento microquirúrgico de la HSA se benefician con la atención protocolizada y la consideración de los resultados del DTC para seleccionar el momento quirúrgico.

Background: Aneurysmal subarachnoid hemorrhage (SAH) surgery, practiced in the first 72 hours is beneficial. The optimal surgical timing, for microsurgical clipping of ruptured intracranial aneurysms, remains controversial when patients arrive between 4 and 14 days. Some surgeons favor a prompt operation regardless the timing. Other ones prefer to wait 2 weeks. Most patients in developing countries are taken to neurosurgical attention late, which not permit an early surgery. Object. To evaluate the surgical outcome in a series of patients with subarachnoid hemorrhage (SAH) managed according to a dynamic protocol. Methods: The authors evaluated surgical outcome by means of Glasgow Outcome Scale (GOS) score in a series of 233 patients with SAH who received neurosurgical clipping in the years 2006-2010 and were followed until January 2012, whose surgical timing was decided according to transcranial Doppler (TD) monitoring. These outcomes were compared with results in a series of 445 historic controls operated 1983-2005. Results: Series mortality at the discharge and at the year were 4.3 and 4.5 percent, and 7 and 7.7 percent in the control group respectively. Series show good outcomes (grade 4 and grade 5 in GOS score) in 93.1 at the discharge and 92.8 percent at the year. Among the historic controls cases with good outcome were 85.6 and 88.1 percent respectively (p = 0.004 y p = 0.036). Conclusions: Surgical outcomes of SAH can be favored by the impact of protocolized attention and TD to decide the best surgical timing in SAH.

Amyloid-beta precursor protein mediates neuronal toxicity of amyloid beta through Go protein activation.

Amyloid beta (Abeta) is a metabolic product of amyloid-beta precursor protein (APP). Deposition of Abeta in the brain and neuronal degeneration are characteristic hallmarks of Alzheimer's disease (AD). Abeta induces neuronal degeneration, but the mechanism of neurotoxicity remains elusive. Here we show that overexpression of APP renders hippocampal neurons vulnerable to Abeta toxicity. Deletion of the extracellular Abeta sequence of APP prevents binding of APP to Abeta, and abolishes toxicity. Abeta toxicity is also abrogated by deletion of the cytoplasmic domain of APP, or by deletions comprising the Go protein-binding sequence of APP. Treatment with Pertussis toxin (PTX) abrogates APP-dependent toxicity of Abeta. Overexpression of PTX-insensitive Galpha-o subunit, but not Galpha-i subunit, of G protein restores Abeta toxicity in the presence of PTX, and this requires the integrity of APP-binding site for Go protein. Altogether, these experiments indicate that interaction of APP with toxic Abeta-species promotes toxicity in hippocampal neurons by a mechanism that involves APP-mediated Go protein activation, revealing an Abeta-receptor-like function of APP directly implicated in neuronal degeneration in AD.

Sex differences and influence of gonadal hormones on MK801-induced neuronal degeneration in the granular retrosplenial cortex of the rat.

MK801, PCP, and ketamine are non-competitive NMDA receptor-antagonists drugs that in humans produce psychomimetic effects and neurocognitive disturbances reminiscent to those of schizophrenia. The administration of these drugs in animals has been used as a pharmacological model to study the NMDA receptor hypofunction-hypothesis of schizophrenia. In animals, the biological effect of MK801 is dose-dependent. Low doses induce behavioral disturbances and higher doses, in addition, promote neurotoxicity in many brain regions, particularly the granular retrosplenial cortex (RSG). The neurotoxic effect of MK801 is sexually dimorphic, being females markedly more sensitive than males; however, the involvement of gonadal hormones is elusive. Here we show that a single intraperitoneal injection of 5 mg/kg of MK801 induced overt neurodegeneration in RSG of female rats, including abundant somatic degeneration in layer 4, and somatodendritic and terminal degeneration in layers 1, 4, and 5. MK801-degeneration in males was scarce and mainly evidenced by the presence of few argirophilic somas in layer 4. Ovariectomized rats were not significantly different than intact females, while orchiectomized rats showed robust MK801-toxicity. Testosterone and dihydrotestosterone (DHT) inhibit MK801-toxicity in orchiectomized rats. In ovariectomized rats only DHT, but not testosterone, prevented MK801-induced degeneration, while in intact females, DHT was only partially protective. Treatment of intact males with estradiol benzoate significantly enhanced MK801-toxicity. Altogether, our experiments indicate that non-aromatizable androgens protect RSG from MK801-toxicity, while estrogens counteract this protection. Thus, the balance of androgens and estrogens delineate the sexual dimorphism of the RSG to the toxic effect of MK801.

Deposition of amyloid fibrils promotes cell-surface accumulation of amyloid beta precursor protein.

Amyloid beta protein (Abeta) deposition and neuronal degeneration are characteristic pathological features of Alzheimer's disease (AD). In vitro, Abeta fibrils (fAbeta) induce neuronal degeneration reminiscent to AD, but the mechanism of neurotoxicity is unknown. Here we show that amyloid fibrils increase the level of cell-surface full-length amyloid beta precursor protein (h-AbetaPP) and secreted AbetaPP (s-AbetaPP). Pulse-chase analysis indicated that fAbeta selectively inhibited the turnover of cell-surface AbetaPP, without altering its intracellular levels. FAbeta-induced AbetaPP accumulation was not abrogated by cycloheximide, suggesting that increased protein synthesis is not critically required. Abeta fibrils sequester s-AbetaPP from the culture medium and promote its accumulation at the cell surface, indicating that binding of Abeta fibrils mediates AbetaPP accumulation. A time course analysis of Abeta treatment showed that AbetaPP level is elevated before significant cell death can be detected, while other toxic insults do not augment AbetaPP level, suggesting that AbetaPP may be specifically involved in early stages of Abeta-induced neurodegeneration. Finally, Abeta fibrils promote clustering of h-AbetaPP in abnormal focal adhesion-like (FA-like) structures that mediate neuronal dystrophy, increasing its association with the cytoskeleton. These results indicate that the interaction of Abeta fibrils with AbetaPP is an early event in the mechanism of Abeta-induced neurodegeneration that may play a significant role in AD pathogenesis.