ABSTRACT
Topotecan is a topoisomerase 1 (TOP1) inhibitor that is used to treat various forms of cancer. We recently found that topotecan reduces the expression of multiple long genes, including many neuronal genes linked to synapses and autism. However, whether topotecan alters synaptic protein levels and synapse function is currently unknown. Here we report that in primary cortical neurons, topotecan depleted synaptic proteins that are encoded by extremely long genes, including Neurexin-1, Neuroligin-1, Cntnap2, and GABA(A)ß3. Topotecan also suppressed spontaneous network activity without affecting resting membrane potential, action potential threshold, or neuron health. Topotecan strongly suppressed inhibitory neurotransmission via pre- and postsynaptic mechanisms and reduced excitatory neurotransmission. The effects on synaptic protein levels and inhibitory neurotransmission were fully reversible upon drug washout. Collectively, our findings suggest that TOP1 controls the levels of multiple synaptic proteins and is required for normal excitatory and inhibitory synaptic transmission.
Subject(s)
DNA Topoisomerases, Type I/metabolism , Excitatory Postsynaptic Potentials/drug effects , Neurons/drug effects , Synapses/drug effects , Topotecan/pharmacology , Action Potentials/drug effects , Animals , Calcium-Binding Proteins , Cell Adhesion Molecules, Neuronal/metabolism , Cells, Cultured , Cerebral Cortex/cytology , Excitatory Postsynaptic Potentials/physiology , Female , Immunoblotting , Mice, Inbred C57BL , Microscopy, Fluorescence , Neural Cell Adhesion Molecules/metabolism , Neurons/metabolism , Neurons/physiology , Patch-Clamp Techniques , Synapses/physiology , Topoisomerase I Inhibitors/pharmacologyABSTRACT
Thiamine monophosphatase (TMPase, also known as fluoride-resistant acid phosphatase) is a classic histochemical marker of small-diameter dorsal root ganglia neurons. The molecular identity of TMPase is currently unknown. We found that TMPase is identical to the transmembrane isoform of prostatic acid phosphatase (PAP), an enzyme with unknown molecular and physiological functions. We then found that PAP knockout mice have normal acute pain sensitivity but enhanced sensitivity in chronic inflammatory and neuropathic pain models. In gain-of-function studies, intraspinal injection of PAP protein has potent antinociceptive, antihyperalgesic, and antiallodynic effects that last longer than the opioid analgesic morphine. PAP suppresses pain by functioning as an ecto-5'-nucleotidase. Specifically, PAP dephosphorylates extracellular adenosine monophosphate (AMP) to adenosine and activates A1-adenosine receptors in dorsal spinal cord. Our studies reveal molecular and physiological functions for PAP in purine nucleotide metabolism and nociception and suggest a novel use for PAP in the treatment of chronic pain.
Subject(s)
5'-Nucleotidase/physiology , Adenosine/biosynthesis , Pain/enzymology , Pain/prevention & control , Protein Tyrosine Phosphatases/physiology , 5'-Nucleotidase/biosynthesis , 5'-Nucleotidase/genetics , Acid Phosphatase , Adenosine/genetics , Adenosine/physiology , Animals , Ganglia, Spinal/cytology , Ganglia, Spinal/metabolism , Ganglia, Spinal/physiology , Humans , Isoenzymes/biosynthesis , Isoenzymes/genetics , Isoenzymes/metabolism , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Pain/genetics , Pain Measurement/methods , Protein Tyrosine Phosphatases/biosynthesis , Protein Tyrosine Phosphatases/genetics , Protein Tyrosine Phosphatases/metabolism , Receptor, Adenosine A1/metabolism , Receptor, Adenosine A1/physiologyABSTRACT
The anionic porphyrin, meso-tetrakis(4-sulfonatophenyl)porphine, is found to tightly bind to an engineered 14-residue peptide, resulting in induced alpha-helix formation when mixed in aqueous solutions. The small porphyrin-peptide dissociation constant (2 muM) observed is related to the energetics of peptide helix formation coupled with electrostatic interactions between the anionic porphyrin and cationic residues in the coiled peptide. Analytical ultracentrifugation measurements indicate the porphyrin-peptide complexes dimerize, probably into a coiled coil, and weakly associate to form even higher order structures.