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1.
J Med Chem ; 54(24): 8299-304, 2011 Dec 22.
Article in English | MEDLINE | ID: mdl-22054058

ABSTRACT

Eight monomeric congeners, related to the multitarget lead candidate memoquin, were prepared and evaluated at multiple targets to determine their profile against Alzheimer's disease. 2-4 bind to AChE with similar low nanomolar affinities and function as effective inhibitors of amyloid aggregation. The most potent monovalent ligand 2 also inhibits BACE-1 in vitro and APP metabolism in primary chicken telencephalic neurons.


Subject(s)
Alkanes/chemical synthesis , Ethylamines/chemical synthesis , Acetylcholinesterase/chemistry , Alkanes/chemistry , Alkanes/pharmacology , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid beta-Peptides/chemistry , Amyloid beta-Peptides/metabolism , Animals , Butyrylcholinesterase/chemistry , Catalytic Domain , Cell Survival/drug effects , Chickens , Cholinesterase Inhibitors/chemical synthesis , Cholinesterase Inhibitors/chemistry , Cholinesterase Inhibitors/pharmacology , Ethylamines/chemistry , Ethylamines/pharmacology , Humans , In Vitro Techniques , Ligands , Models, Molecular , Molecular Conformation , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Binding , Structure-Activity Relationship
2.
J Mol Neurosci ; 43(3): 257-67, 2011 Mar.
Article in English | MEDLINE | ID: mdl-20603724

ABSTRACT

Sequential cleavage of amyloid precursor protein (APP) by ß- and γ-secretases and the formation of Aß peptides are pivotal for Alzheimer's disease. Therefore, a large number of drugs has been developed targeting APP metabolism. However, many pharmacological compounds have been identified in vitro in immortalized APP overexpressing cell lines rather than in primary neurons. Here, we compared the effect of already characterized secretase inhibitors and modulators on Aß formation in primary chicken telencephalic neurons and in a human neuroglioma cell line (H4) ectopically expressing human APP with the Swedish double mutation. Primary chicken neurons replicated the effects of a ß-secretase inhibitor (ß-secretase inhibitor IV), two γ-secretase inhibitors (DAPM, DAPT), two non-steroidal-anti-inflammatory drugs (sulindac sulfide, CW), and of the calpain inhibitor calpeptin. With the exception of the two γ-secretase inhibitors, all tested compounds were more efficacious in primary chicken telencephalic neurons than in the immortalized H4 cell line. Moreover, H4 cells failed to reproduce the effect of calpeptin. Hence, primary chicken telencephalic neurons represent a suitable cell culture model for testing drugs interfering with APP processing and are overall more sensitive to pharmacological interference than immortalized H4 cells ectopically expressing mutant human APP.


Subject(s)
Amyloid beta-Protein Precursor/metabolism , Glioma/metabolism , Neurons/metabolism , Telencephalon/cytology , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/metabolism , Amyloid beta-Peptides/genetics , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/genetics , Animals , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Aspartic Acid Endopeptidases/antagonists & inhibitors , Aspartic Acid Endopeptidases/metabolism , Calpain/metabolism , Cell Line, Tumor , Cells, Cultured , Chickens , Culture Media, Conditioned/chemistry , Humans , Models, Animal , Mutation , Neurons/cytology , Neurons/drug effects , Telencephalon/metabolism
3.
Exp Neurol ; 225(1): 60-73, 2010 Sep.
Article in English | MEDLINE | ID: mdl-20451518

ABSTRACT

Tenascin-C (Tnc) is a multimodular extracellular matrix glycoprotein that is markedly upregulated in CNS injuries where it is primarily secreted by reactive astrocytes. Different Tnc isoforms can be generated by the insertion of variable combinations of one to seven (in rats) alternatively spliced distinct fibronectin type III (FnIII) domains to the smallest variant. Each spliced FnIII repeat mediates specific actions on neurite outgrowth, neuron migration or adhesion. Hence, different Tnc isoforms might differentially influence CNS repair. We explored the expression pattern of Tnc variants after cortical lesions and after treatment of astrocytes with various cytokines. Using RT-PCR, we observed a strong upregulation of Tnc transcripts containing the spliced FnIII domains B or D in injured tissue at 2-4 days post-lesion (dpl). Looking at specific combinations, we showed a dramatic increase of Tnc isoforms harboring the neurite outgrowth-promoting BD repeat with both the B and D domains being adjacent to each other. Isoforms containing only the axon growth-stimulating spliced domain D were also dramatically enhanced after injury. Injury-induced increase of Tnc proteins comprising the domain D was confirmed by Western Blotting and immunostaining of cortical lesions. In contrast, the FnIII modules C and AD1 were weakly modulated after injury. The growth cone repulsive A1A2A4 domains were poorly expressed in normal and injured tissue but the smallest isoform, which is also repellant, was highly expressed after injury. Expression of the shortest Tnc isoform and of variants containing B, D or BD, was strongly upregulated in cultured astrocytes after TGFbeta1 treatment, suggesting that TGFbeta1 could mediate, at least in part, the injury-induced upregulation of these isoforms. We identified complex injury-induced differential regulations of Tnc isoforms that may well influence axonal regeneration and repair processes in the damaged CNS.


Subject(s)
Astrocytes/metabolism , Brain Injuries/metabolism , Fibronectins/metabolism , Tenascin/metabolism , Alternative Splicing/genetics , Animals , Animals, Newborn , Astrocytes/pathology , Brain Injuries/genetics , Brain Injuries/pathology , Cell Adhesion Molecules, Neuronal/biosynthesis , Cell Adhesion Molecules, Neuronal/metabolism , Cell Adhesion Molecules, Neuronal/physiology , Cells, Cultured , Contactins , Disease Models, Animal , Female , Fibronectins/genetics , Fibronectins/physiology , Nerve Regeneration/physiology , Protein Isoforms/biosynthesis , Protein Isoforms/chemistry , Protein Isoforms/physiology , Protein Structure, Tertiary/genetics , Protein Structure, Tertiary/physiology , Rats , Rats, Sprague-Dawley , Tenascin/genetics , Tenascin/physiology
4.
J Neurosci ; 29(17): 5546-57, 2009 Apr 29.
Article in English | MEDLINE | ID: mdl-19403822

ABSTRACT

Damaged CNS axons are prevented from regenerating by an environment containing many inhibitory factors. They also lack an integrin that interacts with tenascin-C, the main extracellular matrix glycoprotein of the CNS, which is upregulated after injury. The alpha9beta1 integrin heterodimer is a receptor for the nonalternatively spliced region of tenascin-C, but the alpha9 subunit is absent in adult neurons. In this study, we show that PC12 cells and adult rat dorsal root ganglion (DRG) neurons do not extend neurites on tenascin-C. However, after forced expression of alpha9 integrin, extensive neurite outgrowth from PC12 cells and adult rat DRG neurons occurs. Moreover, both DRG neurons and PC12 cells secrete tenascin-C, enabling alpha9-transfected cells to grow axons on tissue culture plastic. Using adeno-associated viruses to express alpha9 integrin in vivo in DRGs, we examined axonal regeneration after cervical dorsal rhizotomy or dorsal column crush in the adult rat. After rhizotomy, significantly more dorsal root axons regrew into the dorsal root entry zone at 6 weeks after injury in alpha9 integrin-expressing animals than in green fluorescent protein (GFP) controls. Similarly, after a dorsal column crush injury, there was significantly more axonal growth into the lesion site compared with GFP controls at 6 weeks after injury. Behavioral analysis after spinal cord injury revealed that both experimental and control groups had an increased withdrawal latency in response to mechanical stimulation when compared with sham controls; however, in response to heat stimulation, normal withdrawal latencies returned after alpha9 integrin treatment but remained elevated in control groups.


Subject(s)
Axons/metabolism , Integrin alpha Chains/physiology , Nerve Regeneration/physiology , Neurites/physiology , Sensory Receptor Cells/metabolism , Tenascin/metabolism , Animals , Axons/ultrastructure , Cells, Cultured , Ganglia, Spinal/physiology , Ganglia, Spinal/ultrastructure , Humans , Integrin alpha Chains/biosynthesis , Integrin alpha Chains/genetics , Male , Microscopy, Confocal , Neurites/ultrastructure , Neurogenesis/physiology , PC12 Cells , Rats , Rats, Sprague-Dawley , Sensory Receptor Cells/physiology , Sensory Receptor Cells/ultrastructure
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