Candidate drugs for restoring visual function in blindness
We are using opto-pharmacology to bestow light-sensitivity on neurons without requiring genetic manipulation (Fortin et al., Nature Methods 2008). The most exciting application of this technology is as a potential treatment for blindness (Polosukhina et al. Neuron 2012; Tochitsky et al., 2014). Retinitis pigmentosa (RP) and age-related macular degeneration (AMD) are degenerative blinding diseases caused by the death of rods and cones in the retina, leaving the remainder of the visual system intact but unable to respond to light. Photoswitches can restore light sensitivity to the retina and behavioral responses to living mice afflicted with RP. Intra-ocular injection of photoswitches can restores the pupillary light reflex and visual learning in mice lacking retinal photoreceptors, indicating reconstitution of light signaling through brain circuits. Ongoing studies are aimed at identifying the optimal photoswitch that provides safe, effective, and long-lasting vision restoration.
Rod/cone degeneration opens a natural portal for delivering photoswitches to the blind retina
Our photoswitches have a surprising feature: they are effective in photosensitizing blind retinas in which the rods and cones have died, but it has no effect on healthy retinas that still possess intact rods and cones. This indicates that changes in the entry or action of photoswitches must occur when photoreceptors degenerate. We have found enhanced gene expression of the P2X receptor, a large-conductance ionotropic receptor for ATP. We find that these P2X receptors are up-regulated and tonically active in degenerating retina, and this activity is necessary for the entry of photoswitches into retinal ganglion cells (RGCs). The P2X receptors also allow fluorescent dyes to accumulate and label the same cells (see Figure). Hence we have stumbled upon a naturally drug delivery pathway specific to degeneration. Ongoing studies are aimed at determining the signal triggered by rod/cone degeneration that initiates the up-regulation of P2X receptors.
The degeneration-specific action of photoswitches raises the possibility that they may act locally in restoring light-sensitivity to a partially-damaged retina, while sparing regions that remain healthy. This is a particularly relevant for age-related macular degeneration (AMD), a highly prevalent disorder, characterized by loss of rods and cones only in the macula, the central 2% of the retina responsible for high-acuity vision. Another surprise is that photosensitization is selective for one type of retinal ganglion cell (the OFF-center RGC), rather than indiscriminately photosensitizing all RGCs (Tochitsky et al., Neuron 2016).
Photoswitches for optical control of pain
We are using a photoswitch named QAQ as a light-sensitive analgesic (Mourot et al., Nature Methods 2011). In its trans configuration, QAQ blocks every type of voltage-gated ion channel that we have tested (K+ channels, Na+ channels, Ca2+ channels, and HCN channels). The net effect is to silence neuronal activity in a light-reversible manner. However, QAQ is membrane-impermeant and only affects ion channels when added to the cytoplasmic side of the membrane. We have QAQ can be into cells through large-pore ion channels, including the heat-sensitive capsaicin receptor (TRPV1) and a receptor for ATP (the P2X receptor). TRPV1 channels and P2X receptors are highly expressed in a subset of pain-sensing neurons (nociceptive cells) in the dorsal root ganglion. We can selectively impart light-sensitivity on just these neurons by treating tissue by adding the complementary agonist (i.e. capsaicin or ATP) plus QAQ. Experiments in vivo show that QAQ can be used as a light-regulated analgesic to modulate behavioral responses to noxious stimuli, a finding that has both scientific and clinical implications. Ongoing studies are using QAQ to reveal that nociceptive neurons within the dorsal root ganglia can signal to one another, especially after peripheral nerve injury. Our results suggest that this sensory cross-talk contributes to chronic pain. These experiments are being carried out in collaboration with Frederic Nagy, Professor at the University of Bordeaux, and Dr. Allan Basbaum, Professor at UCSF.
Probing the chronic activity of nociceptive ion channel in peripheral sensory terminals, cell bodies, and central nerve terminals with QAQ (pronounced “quack”, a photoswitch compound that enters cells and photosensitizes action potential firing.
Photoswitches for local control of excitability in parts of a neuron
In the classical view, dendrites are passive recipients of synaptic input signals, but we now know that dendrites in many neurons have voltage-gated ion channels and are electrically excitable. But understanding how dendritic excitability affects synaptic integration and plasticity has been hindered by the small fiber diameter and spatial complexity of dendritic trees. We are using QAQ, DENAQ, and other photoswitch compounds to enable local regulation of excitability in individual dendrites, axons, or presynaptic terminals of neurons, in auditory nuclei, hippocampal neurons, and in retina (Ko et al., Nature Neuroscience 2016). By injecting a photoswich into an individual neuron with a patch electrode, we can optically regulate voltage-gated channels in individual subcellular regions, either putting excitability to sleep or waking it up with different wavelengths of light. Electrical recording and optical imaging of dendrites, axons, or terminals is difficult, but photoswitches provide an easy way to explore the occurrence and functional consequences of dendritic excitability in all sorts of neurons, including many that were previously inaccessible to electrophysiological manipulation.
Intracellular injection of the photoswitch QAQ enables optical control of excitability in brain neurons. Responses to depolarizing and hyperpolarizing current injections. Violet is in 380 nm light (V-gated channels unblocked), green is in 500 nm light (V-gated channels blocked).