María J. Benítez

Live-cell fluorescence imaging reveals the dynamics of protein kinase CK2 individual subunits.

ABSTRACT Protein kinase CK2 is a multifunctional enzyme which has long been described as a stable heterotetrameric complex resulting from the association of two catalytic (α or α′) and two regulatory (β) subunits. To track the spatiotemporal dynamics of CK2 in living cells, we fused its catalytic α and regulatory β subunits with green fluorescent protein (GFP). Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Imaging of stable cell lines expressing low levels of GFP-CK2α or GFP-CK2β revealed the existence of CK2 subunit subpopulations exhibiting differential dynamics. Once in the nucleus, they diffuse randomly at different rates. Unlike CK2β, CK2α can shuttle, showing the dynamic nature of the nucleocytoplasmic trafficking of the kinase. When microinjected in the cytoplasm, the isolated CK2 subunits are rapidly translocated into the nucleus, whereas the holoenzyme complex remains in this cell compartment, suggesting an intramolecular masking of the nuclear localization sequences that suppresses nuclear accumulation. However, binding of FGF-2 to the holoenzyme triggers its nuclear translocation. Since the substrate specificity of CK2α is dramatically changed by its association with CK2β, the control of the nucleocytoplasmic distribution of each subunit may represent a unique potential regulatory mechanism for CK2 activity.

Adenylate cyclase 5 coordinates the action of ADP, P2Y1, P2Y13 and ATP-gated P2X7 receptors on axonal elongation

In adult brains, ionotropic or metabotropic purinergic receptors are widely expressed in neurons and glial cells. They play an essential role in inflammation and neurotransmission in response to purines secreted to the extracellular medium. Recent studies have demonstrated a role for purinergic receptors in proliferation and differentiation of neural stem cells although little is known about their role in regulating the initial neuronal development and axon elongation. The objective of our study was to investigate the role of some different types of purinergic receptors, P2Y1, P2Y13 and P2X7, which are activated by ADP or ATP. To study the role and crosstalk of P2Y1, P2Y13 and P2X7 purinergic receptors in axonal elongation, we treated neurons with specific agonists and antagonists, and we nucleofected neurons with expression or shRNA plasmids. ADP and P2Y1–GFP expression improved axonal elongation; conversely, P2Y13 and ATP-gated P2X7 receptors halted axonal elongation. Signaling through each of these receptor types was coordinated by adenylate cyclase 5. In neurons nucleofected with a cAMP FRET biosensor (ICUE3), addition of ADP or Blue Brilliant G, a P2X7 antagonist, increased cAMP levels in the distal region of the axon. Adenylate cyclase 5 inhibition or suppression impaired these cAMP increments. In conclusion, our results demonstrate a crosstalk between two metabotropic and one ionotropic purinergic receptor that regulates cAMP levels through adenylate cyclase 5 and modulates axonal elongation triggered by neurotropic factors and the PI3K–Akt–GSK3 pathway.

Visualization and molecular analysis of nuclear import of protein kinase CK2 subunits in living cells

We have generated fusion proteins between the subunits of CK2 and GFP and characterized their behaviour in living cells. The expressed fusion proteins were functional and interacted with endogenous CK2. Imaging of NIH3T3 cells expressing low level of GFP-CK2α or GFP-CK2β showed that both proteins were mostly nuclear in interphase. Both CK2 subunits contain nuclear localization domains that target them independently to the nucleus. Once in the nucleus, both subunits diffused rapidly in the nucleoplasm. In mitotic cells, CK2 subunits were dispersed throughout the cytoplasm and were not associated to chromatin. Our data are compatible with the idea that each subunit can translocate individually to the nucleus to interact with each other or with important cellular partners. Understanding the molecular mechanisms which regulate the dynamic localization of CK2 subunits will be of central importance. (Mol Cell Biochem 227: 81-90, 2001)

Interaction between Alzheimer's Aβ1-42 Peptide and DNA Detected by Surface Plasmon Resonance

Alzheimers disease is a form of senile mental disorder characterized by the presence of extracellular plaques, containing amyloid-beta (Abeta) as the main component. According to the amyloid hypothesis, an increase of extracellular Abeta production is in the origin of the aberrant plaques causing neuronal loss and dementia. However, a wealth of evidence has been accumulated pointing to the toxicity of soluble intracellular Abeta, having different morphologies of aggregation, as the origin of the neurodegenerative process. The exact nature of the initial molecular events by which Abeta exerts its neurotoxicity, remains obscure. Different forms of soluble Abeta peptide aggregates have been recently found to reside in the nucleus of CHO cells and Alzheimers disease brain samples. This paper focus mainly on the interaction between DNA and the 42 residue Abeta (Abeta42) as studied by Surface Plasmon Resonance. Electronic microscopy and UV-visible spectroscopy are also used to further characterize the interaction. Particular attention is paid to the extent of Abeta42 aggregation needed to observe the interaction with DNA. Our results show that DNA binds all soluble aggregate forms of Abeta42, therefore suggesting that DNA binding is a general property of different soluble forms of Abeta42, unrelated to the extent of aggregation.

Tau aggregation followed by atomic force microscopy and surface plasmon resonance, and single molecule tau-tau interaction probed by atomic force spectroscopy.

Intracellular neurofibrillary tangles, composed mainly of tau protein, and extracellular plaques, containing mostly amyloid-beta, are the two types of protein aggregates found upon autopsy within the brain of Alzheimers disease patients. Polymers of tau protein can also be found in other neurodegenerative disorders known as tauopathies. Tau is a highly soluble protein, intrinsically devoid of secondary or tertiary structure, as many others proteins particularly prone to form fibrillar aggregations. The mechanism by which this unfolded molecule evolves to the well ordered helical filaments has been amply studied. In fact, it is a very slow process when followed in the absence of aggregation inducers. Herein we describe the use of surface plasmon resonance, atomic force microscopy, and atomic force spectroscopy to detect tau-tau interactions and to follow the process of aggregation in the absence of aggregation inducers. Tau-tau interactions are clearly detected, although a very long period of time is needed to observe filaments formation. Tau oligomers showing a granular appearance, however, are observed immediately as a consequence of this interaction. These granular tau oligomers slowly evolve to larger structures and eventually to filaments having a size smaller than those reported for paired helical filaments purified from Alzheimers disease.

Interaction of Alzheimer's disease amyloid β peptide fragment 25–35 with tau protein, and with a tau peptide containing the microtubule binding domain

The interaction of amyloid beta (Abeta) 25-35 with tau protein and with the peptide 1/2R (KVTSKCGSLGNIHHKPGGG), has been investigated by chromatography, electron microscopy, and surface plasmon resonance (SPR). Abeta 25-35 comprises the minimum region of Abeta peptide that is able to aggregate into fibrils, and 1/2R contains residues 307-325 from the tau region involved in microtubule binding. The results of chromatography showed that Abeta 25-35 induces the aggregation of tau protein and of tau peptide 1/2R. Likewise, the results of electron microscopy showed that Abeta 25-35 increases the tau peptide polymerization observed in the presence of polyanions like heparin. A decrease in Abeta 25-35 aggregation induced by tau peptide was also observed by both techniques. No direct interaction between tau protein immobilized on the sensor surface and Abeta 25-35 could be detected by SPR. However, incubation of tau protein at room temperature produced the loss of capability of this protein for interacting with the active biosensor surface. The presence of Abeta 25-35 during the incubation of tau protein makes more efficient this loss of interacting capability with the sensor surface. These results clearly indicate that Abeta 25-35, the peptide region to which the cytotoxic properties of Abeta can be assigned, interacts with the peptide region of tau protein involved in microtubule binding. This interaction produces the aggregation of tau peptide and the concomitant disassembling of Abeta 25-35, offering thus an explanation to the lack of co-localization of neurofibrillary tangles and senile plaques in Alzheimers disease, and suggesting the possibility that tau protein may have a protective action by preventing Abeta from adopting the cytotoxic, aggregated form.

Thermodynamics of the Interaction between Alzheimer's Disease Related Tau Protein and DNA

Tau hyperphosphorylation can be considered as one of the hallmarks of Alzheimers disease and other tauophaties. Besides its well-known role as a microtubule associated protein, Tau displays a key function as a protector of genomic integrity in stress situations. Phosphorylation has been proven to regulate multiple processes including nuclear translocation of Tau. In this contribution, we are addressing the physicochemical nature of DNA-Tau interaction including the plausible influence of phosphorylation. By means of surface plasmon resonance (SPR) we measured the equilibrium constant and the free energy, enthalpy and entropy changes associated to the Tau-DNA complex formation. Our results show that unphosphorylated Tau binding to DNA is reversible. This fact is in agreement with the protective role attributed to nuclear Tau, which stops binding to DNA once the insult is over. According to our thermodynamic data, oscillations in the concentration of dephosphorylated Tau available to DNA must be the variable determining the extent of Tau binding and DNA protection. In addition, thermodynamics of the interaction suggest that hydrophobicity must represent an important contribution to the stability of the Tau-DNA complex. SPR results together with those from Tau expression in HEK cells show that phosphorylation induces changes in Tau protein which prevent it from binding to DNA. The phosphorylation-dependent regulation of DNA binding is analogous to the Tau-microtubules binding inhibition induced by phosphorylation. Our results suggest that hydrophobicity may control Tau location and DNA interaction and that impairment of this Tau-DNA interaction, due to Tau hyperphosphorylation, could contribute to Alzheimers pathogenesis.

Dysfunction of the PI3K-Akt-GSK-3 pathway is a common feature in cell culture and in vivo models of prion disease.

Transmissible spongiform encephalopathies, also called prion diseases, are characterized by the cerebral accumulation of misfolded prion protein (PrPSC) and subsequent neurodegeneration. However, despite considerable research effort, the molecular mechanisms underlying prion‐induced neurodegeneration are poorly understood. Here, we explore the hypothesis that prions induce dysfunction of the PI3K/Akt/GSK‐3 signalling pathway.

LGI1 tunes intrinsic excitability by regulating the density of axonal Kv1 channels

Significance Leucine-rich glioma-inactivated 1 (LGI1) is a secreted neuronal protein associated in a multiprotein complex with Kv1 channels. Natural mutations in this protein are found in the early-adulthood–onset disorder “autosomal dominant epilepsy with auditory features” and animal models with Lgi1 genetic deletion display spontaneous seizures. We investigated the potential link between LGI1 depletion and intrinsic neuronal excitability. We found that LGI1 determines neuronal excitability in CA3 pyramidal neurons through the control of axonal Kv1-channel expression. Genetic deletion of Lgi1 induces a robust diminution in Kv1 channels, especially in axons where they colocalize with LGI1, very likely contributing to epileptogenesis. These results strongly suggest that LGI1 controls neuronal excitability by regulating Kv1-channel expression. Autosomal dominant epilepsy with auditory features results from mutations in leucine-rich glioma-inactivated 1 (LGI1), a soluble glycoprotein secreted by neurons. Animal models of LGI1 depletion display spontaneous seizures, however, the function of LGI1 and the mechanisms by which deficiency leads to epilepsy are unknown. We investigated the effects of pure recombinant LGI1 and genetic depletion on intrinsic excitability, in the absence of synaptic input, in hippocampal CA3 neurons, a classical focus for epileptogenesis. Our data indicate that LGI1 is expressed at the axonal initial segment and regulates action potential firing by setting the density of the axonal Kv1.1 channels that underlie dendrotoxin-sensitive D-type potassium current. LGI1 deficiency incurs a >50% down-regulation of the expression of Kv1.1 and Kv1.2 via a posttranscriptional mechanism, resulting in a reduction in the capacity of axonal D-type current to limit glutamate release, thus contributing to epileptogenesis.

Tau Protein Provides DNA with Thermodynamic and Structural Features which are Similar to those Found in Histone-DNA Complex

Tau protein has been proposed as a trigger of Alzheimers disease once it is hyperphosphorylated. However, the role that native tau forms play inside the neuronal nucleus remains unclear. In this work we present results concerning the interaction of tau protein with double-stranded DNA, single-stranded DNA, and also with a histone-DNA complex. The tau-DNA interaction results in a structure resembling the beads-on-a-string form produced by the binding of histone to DNA. DNA retardation assays show that tau and histone induce similar DNA retardation. A surface plasmon resonance study of tau-DNA interaction also confirms the minor groove of DNA as a binding site for tau, similarly to the histone binding. A residual binding of tau to DNA in the presence of Distamycin A, a minor groove binder, suggests the possibility that additional structural domains on DNA may be involved in tau interaction. Finally, DNA melting experiments show that, according to the Zipper model of helix-coil transition, the binding of tau increases the possibility of opening the DNA double helix in isolated points along the chain, upon increasing temperature. This behavior is analogous to histones and supports the previously reported idea that tau could play a protective role in stress situations. Taken together, these results show a similar behavior of tau and histone concerning DNA binding, suggesting that post-translational modifications on tau might impair the role that, by modulating the DNA function, might be attributable to the DNA-tau interaction.