Novel Brain-Penetrant, Small-Molecule Tubulin Destabilizers for the Treatment of Glioblastoma.

Glioblastoma (GB) is the most lethal brain cancer in adults, with a 5-year survival rate of 5%. The standard of care for GB includes maximally safe surgical resection, radiation, and temozolomide (TMZ) therapy, but tumor recurrence is inevitable in most GB patients. Here, we describe the development of a blood-brain barrier (BBB)-penetrant tubulin destabilizer, RGN3067, for the treatment of GB. RGN3067 shows good oral bioavailability and achieves high concentrations in rodent brains after oral dosing (Cmax of 7807 ng/mL (20 µM), Tmax at 2 h). RGN3067 binds the colchicine binding site of tubulin and inhibits tubulin polymerization. The compound also suppresses the proliferation of the GB cell lines U87 and LN-18, with IC50s of 117 and 560 nM, respectively. In four patient-derived GB cell lines, the IC50 values for RGN3067 range from 148 to 616 nM. Finally, in a patient-derived xenograft (PDX) mouse model, RGN3067 reduces the rate of tumor growth compared to the control. Collectively, we show that RGN3067 is a BBB-penetrant small molecule that shows in vitro and in vivo efficacy and that its design addresses many of the physicochemical properties that prevent the use of microtubule destabilizers as treatments for GB and other brain cancers.

Cul4 ubiquitin ligase cofactor DCAF12 promotes neurotransmitter release and homeostatic plasticity.

We genetically characterized the synaptic role of the Drosophila homologue of human DCAF12, a putative cofactor of Cullin4 (Cul4) ubiquitin ligase complexes. Deletion of Drosophila DCAF12 impairs larval locomotion and arrests development. At larval neuromuscular junctions (NMJs), DCAF12 is expressed presynaptically in synaptic boutons, axons, and nuclei of motor neurons. Postsynaptically, DCAF12 is expressed in muscle nuclei and facilitates Cul4-dependent ubiquitination. Genetic experiments identified several mechanistically independent functions of DCAF12 at larval NMJs. First, presynaptic DCAF12 promotes evoked neurotransmitter release. Second, postsynaptic DCAF12 negatively controls the synaptic levels of the glutamate receptor subunits GluRIIA, GluRIIC, and GluRIID. The down-regulation of synaptic GluRIIA subunits by nuclear DCAF12 requires Cul4. Third, presynaptic DCAF12 is required for the expression of synaptic homeostatic potentiation. We suggest that DCAF12 and Cul4 are critical for normal synaptic function and plasticity at larval NMJs.

Mitochondria on the Road to Power Axonal Regeneration.

In this issue of Neuron, Han et al. (2016) and Cartoni et al. (2016) define a critical role of mitochondrial transport for successful axon regeneration after injury and provide new insights into intrinsic mechanisms controlling neuronal regeneration capacity in worms and mice.

Chemoselective tarantula toxins report voltage activation of wild-type ion channels in live cells.

Electrically excitable cells, such as neurons, exhibit tremendous diversity in their firing patterns, a consequence of the complex collection of ion channels present in any specific cell. Although numerous methods are capable of measuring cellular electrical signals, understanding which types of ion channels give rise to these signals remains a significant challenge. Here, we describe exogenous probes which use a novel mechanism to report activity of voltage-gated channels. We have synthesized chemoselective derivatives of the tarantula toxin guangxitoxin-1E (GxTX), an inhibitory cystine knot peptide that binds selectively to Kv2-type voltage gated potassium channels. We find that voltage activation of Kv2.1 channels triggers GxTX dissociation, and thus GxTX binding dynamically marks Kv2 activation. We identify GxTX residues that can be replaced by thiol- or alkyne-bearing amino acids, without disrupting toxin folding or activity, and chemoselectively ligate fluorophores or affinity probes to these sites. We find that GxTX-fluorophore conjugates colocalize with Kv2.1 clusters in live cells and are released from channels activated by voltage stimuli. Kv2.1 activation can be detected with concentrations of probe that have a trivial impact on cellular currents. Chemoselective GxTX mutants conjugated to dendrimeric beads likewise bind live cells expressing Kv2.1, and the beads are released by channel activation. These optical sensors of conformational change are prototype probes that can indicate when ion channels contribute to electrical signaling.

Reduced gamma frequency in the medial frontal cortex of aged rats during behavior and rest: implications for age-related behavioral slowing.

Age-related cognitive and behavioral slowing may be caused by changes in the speed of neural signaling or by changes in the number of signaling steps necessary to achieve a given function. In the mammalian cortex, neural communication is organized by a 30-100 Hz "gamma" oscillation. There is a putative link between the gamma frequency and the speed of processing in a neural network: the dynamics of pyramidal neuron membrane time constants suggest that synaptic integration is framed by the gamma cycle, and pharmacological slowing of gamma also slows reaction times on behavioral tasks. The present experiments identify reductions in a robust 40-70 Hz gamma oscillation in the aged rat medial frontal cortex. The reductions were observed in the form of local field potentials, later peaks in fast-spiking neuron autocorrelations, and delays in the spiking of inhibitory neurons following local excitatory signals. Gamma frequency did not vary with movement speed, but rats with slower gamma also moved more slowly. Gamma frequency age differences were not observed in hippocampus. Hippocampal CA1 fast-spiking neurons exhibited interspike intervals consistent with a fast (70-100 Hz) gamma frequency, a pattern maintained across theta phases and theta frequencies independent of fluctuations in the average firing rates of the neurons. We propose that an average lengthening of the cortical 15-25 ms gamma cycle is one factor contributing to age-related slowing and that future attempts to offset cognitive declines will find a target in the response of fast-spiking inhibitory neurons to excitatory inputs.