benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and Neurodegenerative-Diseases

Chronic neurodegenerative syndromes such as Alzheimer's and Parkinson's diseases, or acute syndromes such as ischemic stroke or traumatic brain injuries are characterized by early synaptic collapse which precedes axonal and neuronal cell body degeneration and promotes early cognitive impairment in patients. Until now, neuroprotective strategies have failed to impede the progression of neurodegenerative syndromes. Drugs preventing the loss of cell body do not prevent the cognitive decline, probably because they lack synapto-protective effects. The absence of physiologically realistic neuronal network models which can be easily handled has hindered the development of synapto-protective drugs suitable for therapies. Here we describe a new microfluidic platform which makes it possible to study the consequences of axonal trauma of reconstructed oriented mouse neuronal networks. Each neuronal population and sub-compartment can be chemically addressed individually. The somatic, mid axon, presynaptic and postsynaptic effects of local pathological stresses or putative protective molecules can thus be evaluated with the help of this versatile "brain on chip" platform. We show that presynaptic loss is the earliest event observed following axotomy of cortical fibers, before any sign of axonal fragmentation or post-synaptic spine alteration. This platform can be used to screen and evaluate the synapto-protective potential of several drugs. For instance, NAD⁺ and the Rho-kinase inhibitor Y27632 can efficiently prevent synaptic disconnection, whereas the broad-spectrum caspase inhibitor zVAD-fmk and the stilbenoid resveratrol do not prevent presynaptic degeneration. Hence, this platform is a promising tool for fundamental research in the field of developmental and neurodegenerative neurosciences, and also offers the opportunity to set up pharmacological screening of axon-protective and synapto-protective drugs.

Topics: Amides; Amino Acid Chloromethyl Ketones; Animals; Axons; Dendrites; Embryo, Mammalian; Enzyme Inhibitors; Mice; Microfluidics; Microscopy, Fluorescence; Models, Neurological; NAD; Nerve Net; Neurodegenerative Diseases; Primary Cell Culture; Pyridines; Resveratrol; Stilbenes; Synapses

Glycogen synthase kinase-3beta (GSK-3beta) is a crucial component in the cascade of events that culminate in a range of neurodegenerative diseases. It is controlled by several pathways, including calpain-mediated cleavage. Calpain mediates in cell death induced by 3-nitropropionic acid (3-NP), but GSK-3beta regulation has not been demonstrated. Here we studied changes in total GSK-3beta protein levels and GSK-3beta phosphorylation at Ser-9 in this model. The 3-NP treatment induced GSK-3beta truncation. This regulation was dependent on calpain activation, since addition of calpeptin to the medium prevented this cleavage. While calpain inhibition prevented 3-NP-induced neuronal loss, inhibition of GSK-3beta by SB-415286 did not. Furthermore, inhibition of cdk5, a known target of calpain involved in 3-NP-induced cell death, also failed to rescue neurons in our model. Our results point to a new target of calpain and indicate possible cross-talk between calpain and GSK-3beta in the 3-NP toxicity pathway. On the basis of our findings, we propose that calpain may modulate 3-NP-induced neuronal loss.

Topics: Amino Acid Chloromethyl Ketones; Aminophenols; Animals; Calpain; Caspases; Cell Survival; Cells, Cultured; Convulsants; Disease Models, Animal; Embryo, Mammalian; Enzyme Inhibitors; Gene Expression Regulation, Enzymologic; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Hippocampus; Male; Maleimides; Mice; Neurodegenerative Diseases; Neurons; Neuroprotective Agents; Nitro Compounds; Propionates; Purines; Rats; Roscovitine; Signal Transduction; Time Factors

Proteasomal dysfunction has been implicated in neurodegenerative disorders and during aging processes. In frontotemporal dementias, corticobasal degeneration, and progressive supranuclear palsy, oligodendrocytes are specifically damaged. Application of proteasomal inhibitors to cultured oligodendrocytes is associated with apoptotic cell death. The present study was undertaken to investigate the death pathway activated in oligodendrocytes by proteasomal inhibition. Our data show that the proteasomal inhibitor MG-132 causes oxidative stress, as indicated by the upregulation of the small heat shock protein heme oxygenase-1 (HO-1) and the appearance of oxidized proteins. Activation of the mitochondrial pathway was involved in the apoptotic process. Mitochondrial membrane potential was disturbed, and cytochrome c was released from the mitochondria. Concomitantly, death-related caspases 3 and 9 were activated and poly(ADP-ribose)-polymerase cleavage occurred. MG-132-induced cell death, DNA-fragmentation, and caspase activation could be prevented by the broad caspase inhibitor zVAD-fmk. In contrast to oligodendrocytes, cultured astrocytes showed resistance to the treatment with proteasomal inhibitors and did not reveal cytotoxic responses. This was also observed in astrocytes differentiated in the presence of dibutyryl cyclic AMP. Hence, individual cells respond differently to proteasomal inhibition and the therapeutic use of proteasomal inhibitors, e.g. for the treatment of cancer or inflammatory diseases, needs to be carefully evaluated.

Topics: Amino Acid Chloromethyl Ketones; Animals; Animals, Newborn; Apoptosis; Astrocytes; Brain; Bucladesine; Caspases; Cells, Cultured; Cysteine Proteinase Inhibitors; Cytochromes c; Enzyme Inhibitors; Heme Oxygenase-1; Leupeptins; Mitochondria; Mitochondrial Membranes; Neurodegenerative Diseases; Oligodendroglia; Oxidative Stress; Poly(ADP-ribose) Polymerases; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Rats; Rats, Wistar

To date, eight neurodegenerative disorders, including Huntington's disease and dentatorubral-pallidoluysian atrophy, have been identified to be caused by expansion of a CAG repeat coding for a polyglutamine (polyQ) stretch. It is, however, unclear how polyQ expansion mediates neuronal cell death observed in these disorders. Here, we have established a tetracycline-regulated expression system producing 19 and 56 repeats of glutamine fused with green fluorescent protein. Induced expression of the 56 polyQ, but not of the 19 polyQ stretch caused marked nuclear aggregation and apoptotic morphological changes of the nucleus. In vitro enzyme assays and Western blotting showed that polyQ56 expression sequentially activated initiator and effector caspases, such as caspase-8 or -9, and caspase-3, respectively. Furthermore, using cell-permeable fluorogenic substrate, the activation of caspase-3-like proteases was demonstrated in intact cells with aggregated polyQ. This is the first direct evidence that the expression of extended polyQ activates caspases and together with the previous findings that some of the products of genes responsible for CAG repeat diseases are substrates of caspase-3 indicates an important role of caspases in the pathogenesis of these diseases.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Blotting, Western; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cell Nucleus; Enzyme Activation; Female; Green Fluorescent Proteins; Humans; Luminescent Proteins; Neurodegenerative Diseases; Oligopeptides; Ovarian Neoplasms; Peptides; Poly(ADP-ribose) Polymerases; Protein Binding; Recombinant Fusion Proteins; Transfection; Tumor Cells, Cultured