Biomedical subjects

Paul De Koninck

Publications and source records attributed to Paul De Koninck.

9 recordsLinked to original sources

PubMed2006

Manipulation of protein kinases reveals different mechanisms for upregulation of heat shock proteins in motor neurons and non-neuronal cells.

Motor neurons have a high threshold for induction of heat shock proteins (Hsps) in response to stress, a property associated with impaired ability to activate heat shock transcription factor 1 (Hsf1). Hyperphosphorylation of Hsf1 has been established as a requirement for transactivation of heat shock genes. This study demonstrated that the impaired heat shock response in motor neurons is not due to altered phosphorylation of Hsf1 by kinases previously shown to affect activation of Hsf1 in other cells (PKC, GSK3beta, ERK1, CaMKIIalpha). However, a constitutively active form of CaMKIV induced robust expression of Hsp70, as well as transcription of a GFP reporter gene driven by the human inducible Hsp70 promoter in unstressed motor neurons, but not in mouse embryonic fibroblasts. The results point to novel mechanisms of activation of heat shock genes in motor neurons that have relevance to exploitation of endogenous stress responses therapeutically.

Animals↗

PubMed2006

Dendritic spine viscoelasticity and soft-glassy nature: balancing dynamic remodeling with structural stability.

Neuronal dendritic spines are a key component of brain circuitry, implicated in many mechanisms for plasticity and long-term stability of synaptic communication. They can undergo rapid actin-based activity-dependent shape fluctuations, an intriguing biophysical property that is believed to alter synaptic transmission. Yet, because of their small size (approximately 1 microm or less) and metastable behavior, spines are inaccessible to most physical measurement techniques. Here we employ atomic force microscopy elasticity mapping and novel dynamic indentation methods to probe the biomechanics of dendritic spines in living neurons. We find that spines exhibit 1), a wide range of rigidities, correlated with morphological characteristics, axonal association, and glutamatergic stimulation, 2), a uniquely large viscosity, four to five times that of other cell types, consistent with a high density of solubilized proteins, and 3), weak power-law rheology, described by the soft-glassy model for cellular mechanics. Our findings provide a new perspective on spine functionality and identify key mechanical properties that govern the ability of spines to rapidly remodel and regulate internal protein trafficking but also maintain structural stability.

Animals↗

PubMed2006

Transition from reversible to persistent binding of CaMKII to postsynaptic sites and NR2B.

Changes in protein-protein interactions and activity states have been proposed to underlie persistent synaptic remodeling that is induced by transient stimuli. Here, we show an unusual stimulus-dependent transition from a short-lived to long-lasting binding between a synaptic receptor and its transducer. Both molecules, the NMDA receptor subunit NR2B and Ca2+/calmodulin (CaM)-dependent protein kinase II (CaMKII), are strongly implicated in mediating synaptic plasticity. We show that CaMKII reversibly translocates to synaptic sites in response to brief stimuli, but its resident time at the synapse increases after longer stimulation. Thus, CaMKII localization reflects temporal patterns of synaptic stimulation. We have identified two surface regions of CaMKII involved in short-lived and long-term interactions with NR2B. Our results support an initial reversible and Ca2+/CaM-dependent binding at the substrate-binding site ("S-site"). On longer stimulation, a persistent interaction is formed at the T286-binding site ("T-site"), thereby keeping the autoregulatory domain displaced and enabling Ca2+/CaM-independent kinase activity. Such dual modes of interaction were observed in vitro and in HEK cells. In hippocampal neurons, short-term stimulation initiates a reversible translocation, but an active history of stimulation beyond some threshold produces a persistent synaptic localization of CaMKII. This activity-dependent incorporation of CaMKII into postsynaptic sites may play a role in maturation and plasticity of synapses.

2-Amino-5-phosphonovalerate↗

PubMed2006

[The fragile X syndrome: one protein missing and 1001 disoriented mRNAs].

Fragile X syndrome is the most common form of inherited mental retardation. This X-linked disease is due to transcriptional silencing of the Fragile Mental Retardation 1 (FMR1) gene and the absence of its gene product, FMRP. This protein is an RNA-binding protein present in mRNP complexes associated with the translation machinery and is thought to be a key player in the control of mRNA transport in neurons. However, the exact role of FMRP in translation remains unclear. Two homologous proteins, FXR1P and FXR2P, are also found in RNP complexes containing FMRP, suggesting that FMRP's functions are much more complex than first thought. The molecular mechanisms altered in cells lacking FMRP still remain to be elucidated, as well as the putative roles of FXR1P and FXR2P as compensatory molecules. Here, we review the various possible functions of FMRP in RNA localization and transport in highly differentiated cells containing dendritic extensions such as neurons.

Fragile X Messenger Ribonucleoprotein 1↗

PubMed2005

Two-photon fluorescent microlithography for live-cell imaging.

Fluorescent dyes added to UV-cure resins allow the rapid fabrication of fluorescent micropatterns on standard glass coverslips by two-photon optical lithography. We use this lithographic method to tailor fiduciary markers, focal references, and calibration tools, for fluorescence and laser scanning microscopy. Fluorescent microlithography provides spatial landmarks to quantify molecular transport, cell growth and migration, and to compensate for focal drift during time-lapse imaging. We show that the fluorescent patterned microstructures are biocompatible with cultures of mammalian cell lines and hippocampal neurons. Furthermore, the high-relief topology of the lithographed substrates is utilized as a mold for poly(dimethylsiloxane) stamps to create protein patterns by microcontact printing, representing an alternative to the current etching techniques. We present two different applications of such protein patterns for localizing cell adhesion and guidance of neurite outgrowth.

Animals↗

PubMed2005

A mechanism for Ca2+/calmodulin-dependent protein kinase II clustering at synaptic and nonsynaptic sites based on self-association.

The activity of Ca2+/calmodulin-dependent protein kinase II (CaMKII) plays an integral role in regulating synaptic development and plasticity. We designed a live-cell-imaging approach to monitor an activity-dependent clustering of green fluorescent protein (GFP)-CaMKII holoenzymes, termed self-association, a process that we hypothesize contributes to the translocation of CaMKII to synaptic and nonsynaptic sites in activated neurons. We show that GFP-CaMKII self-association in human embryonic kidney 293 (HEK293) cells requires a catalytic domain and multimeric structure, requires Ca2+ stimulation and a functional Ca2+/CaM-binding domain, is regulated by cellular pH and Thr286 autophosphorylation, and has variable rates of dissociation depending on Ca2+ levels. Furthermore, we show that the same rules that govern CaMKII self-association in HEK293 cells apply for extrasynaptic and postsynaptic translocation of GFP-CaMKII in hippocampal neurons. Our data support a novel mechanism for targeting CaMKII to postsynaptic sites after neuronal activation. As such, CaMKII may form a scaffold that, in combination with other synaptic proteins, recruits and localizes additional proteins to the postsynaptic density. We discuss the potential function of CaMKII self-association as a tag of synaptic activity.

Animals↗

PubMed2003

Trans-synaptic shift in anion gradient in spinal lamina I neurons as a mechanism of neuropathic pain.

Modern pain-control theory predicts that a loss of inhibition (disinhibition) in the dorsal horn of the spinal cord is a crucial substrate for chronic pain syndromes. However, the nature of the mechanisms that underlie such disinhibition has remained controversial. Here we present evidence for a novel mechanism of disinhibition following peripheral nerve injury. It involves a trans-synaptic reduction in the expression of the potassium-chloride exporter KCC2, and the consequent disruption of anion homeostasis in neurons of lamina I of the superficial dorsal horn, one of the main spinal nociceptive output pathways. In our experiments, the resulting shift in the transmembrane anion gradient caused normally inhibitory anionic synaptic currents to be excitatory, substantially driving up the net excitability of lamina I neurons. Local blockade or knock-down of the spinal KCC2 exporter in intact rats markedly reduced the nociceptive threshold, confirming that the reported disruption of anion homeostasis in lamina I neurons was sufficient to cause neuropathic pain.

Animals↗

PubMed2003

Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations: a simple model.

The rules that govern the activation and autophosphorylation of the multifunctional Ca2+-calmodulin kinase II (CaMKII) by Ca2+ and calmodulin (CaM) are thought to underlie its ability to decode Ca2+ oscillations and to control multiple cellular functions. We propose a simple biophysical model for the activation of CaMKII by Ca2+ and calmodulin. The model describes the transition of the subunits of the kinase between their different possible states (inactive, bound to Ca2+-CaM, phosphorylated at Thr(286), trapped and autonomous). All transitions are described by classical kinetic equations except for the autophosphorylation step, which is modeled in an empirical manner. The model quantitatively reproduces the experimentally demonstrated frequency sensitivity of CaMKII [Science 279 (1998) 227]. We further use the model to investigate the role of several characterized features of the kinase--as well as some that are not easily attainable by experiments--in its frequency-dependent responses. In cellular microdomains, CaMKII is expected to sense very brief Ca2+ spikes; our simulations under such conditions reveal that the enzyme response is tuned to optimal frequencies. This prediction is then confirmed by experimental data. This novel and simple model should help in understanding the rules that govern CaMKII regulation, as well as those involved in decoding intracellular Ca2+ signals.

Biophysical Phenomena↗

PubMed2002

Alternative splicing modulates the frequency-dependent response of CaMKII to Ca(2+) oscillations.

Ca(2+) oscillations are required in various signal trans duction pathways, and contain information both in their amplitude and frequency. Remarkably, the Ca(2+)/calmodulin(CaM)-dependent protein kinase II (CaMKII) can decode such frequencies. A Ca(2+)/CaM-stimulated autophosphorylation leads to Ca(2+)/CaM-independent (autonomous) activity of the kinase that outlasts the initial stimulation. This autonomous activity increases exponentially with the frequency of Ca(2+) oscillations. Here we show that three beta-CaMKII splice variants (beta(M), beta and beta(e)') have very similar specific activity and maximal autonomy. However, their autonomy generated by Ca(2+) oscillations differs significantly. A mechanistic basis was found in alterations of the CaM activation constant and of the initial rate of autophosphorylation. Structurally, the splice variants differ only in a variable 'linker' region between the kinase and association domains. Therefore, we propose that differences in relative positioning of kinase domains within multimeric holoenzymes are responsible for the observed effects. Notably, the beta-CaMKII splice variants are differentially expressed, even among individual hippocampal neurons. Taken together, our results suggest that alternative splicing provides cells with a mechanism to modulate their sensitivity to Ca(2+) oscillations.

Alternative Splicing↗