Alfredo Lorenzo

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.

Amyloid-β precursor protein mediates neuronal toxicity of amyloid β 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 Alzheimers 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.

Wnt-5a/Frizzled9 Receptor Signaling through the Gαo-Gβγ 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 Gβγ 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.

Neurodegeneration and prolonged immediate early gene expression throughout cortical areas of the rat brain following acute administration of dizocilpine.

N-methyl-d-aspartate receptor antagonist drugs (NMDA-A), such as dizocilpine (MK801), induce long-lasting behavioral disturbances reminiscent to psychotic disorders in humans. To identify cortical structures affected by NMDA-A, we used a single dose of MK801 (10 mg/kg) that caused low and high neurodegeneration in intact and orchiectomized male rats, respectively. Degenerating somas (neuronal death) and axonal/synaptic endings (terminal degeneration) were depicted by a silver technique, and functionally affected cortical neuronal subpopulations by Egr-1, c-Fos, and FosB/DeltaFosB-immunolabeling. In intact males, MK801 triggered a c-Fos induction that remained high for more than 24 h in selected layers of the retrosplenial, somatosensory and entorhinal cortices. MK801-induced neurodegeneration reached its peak at 72 h. Degenerating somas were restricted to layer IV of the granular subdivision of the retrosplenial cortex, and were accompanied by suppression of Egr-1 immunolabeling. Terminal degeneration extended to selected layers of the retrosplenial, somatosensory and parahippocampal cortices, which are target areas of retrosplenial cortex. Induction of FosB/DeltaFosB by MK801 also extended to the same cortical layers affected by terminal degeneration, likely reflecting the damage of synaptic connectivity. In orchiectomized males, the neurodegenerative and functional effects of MK801 were exacerbated. Degenerative somas in layer IV of the retrosplenial cortex significantly increased, with a parallel enhancement of terminal degeneration and FosB/DeltaFosB-expression in the mentioned cortical structures, but no additional areas were affected. These observations reveal that synaptic dysfunction/degeneration in the retrosplenial, somatosensory and parahippocampal cortices might underlie the long-lasting impairments induced by NMDA-A.

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

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

The physiological role of the Amyloid Precursor Protein (APP) 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.

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.

Involvement of AMPA/kainate-excitotoxicity in MK801-induced neuronal death in the retrosplenial cortex.

MK801 is a prototypical non-competitive NMDA receptor-antagonist that induces behavioural changes and reversible toxicity at low doses, while at higher doses triggers neuronal death that mainly affects the retrosplenial cortex (RSC) and to a lesser extent other structures such as the posterolateral cortical amygdaloid nucleus (PLCo). The mechanism of MK801-induced neurodegeneration remains poorly understood. In this study we analysed the participation of GABA-ergic and glutamatergic neurotransmission in MK801-induced neuronal death. We used a single i.p. injection of MK801 (2.5 mg/kg) that induced moderate neuronal death in the RSC and PLCo of female rats, and combined this treatment with the i.p., i.c.v., or intra-RSC infusion of drugs that are selective agonists or antagonists of the GABA-ergic or glutamatergic neurotransmission. We found that neuronal death in the RSC, but not the PLCo, was significantly reduced by the i.p. injection of thiopental, and the i.c.v. application of muscimol, both GABA-A agonists. MK801-toxicity in RSC was abrogated by intra-RSC infusion of muscimol, but the GABA antagonist picrotoxin had no effect. HPLC-analysis showed that levels of glutamate, but not GABA, in the RSC decreased after i.p. treatment with MK801. Intra-RSC infusion of MK801 did not enhance toxicity triggered by the i.p. injection of MK801, indicating that toxicity is not due to direct blockade of NMDA receptors in RSC neurons. MK801-toxicity in the RSC was abrogated by i.c.v. and intra-RSC infusions of the AMPA/kainate antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX). Interestingly, i.c.v. application of neither muscimol or DNQX inhibited MK801-toxicity in the PLCo, suggesting that the mechanism of neuronal death in the RSC and the PLCo might be different. 1-naphthylacetyl spermine trihydrochloride (NASPM), which blocks Ca2+ permeable AMPA/kainate receptors, also reduced MK801-induced toxicity in the RSC. Intra-RSC infusion of AMPA or kainic acid alone promoted death of RSC neurons and was reminiscent of the degeneration induced by the i.p. treatment with MK801. Collectively, these experiments provide evidence for an AMPA/kainate-dependent mechanism of excitotoxicity in the death of RSC neurons after i.p. treatment with MK801.

APP/Go protein Gβγ-complex signaling mediates Aβ degeneration and cognitive impairment in Alzheimer's disease models

Deposition of amyloid-β (Aβ), the proteolytic product of the amyloid precursor protein (APP), might cause neurodegeneration and cognitive decline in Alzheimers disease (AD). However, the direct involvement of APP in the mechanism of Aβ-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 Aβ deposition dramatically enhanced APP-Go protein interaction in dystrophic neurites. APP overexpression rendered neurons vulnerable to Aβ toxicity by a mechanism that required Go-Gβγ complex signaling and p38-mitogen-activated protein kinase activation. Gallein, a selective pharmacological inhibitor of Gβγ complex, inhibited Aβ-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 Aβ pathology. Our data provide further evidence for the involvement of APP/Go protein in Aβ-induced degeneration and reveal that Gβγ complex is a signaling target potentially relevant for developing therapies for halting Aβ degeneration in AD.

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.