Dissecting sensory neuron cell diversity
The peripheral nervous system is made up of subpopulations of functionally diverse neuronal types that detect and encode different types of stimuli. In order to understand this functional organization, the Heppenstall laboratory has developed a number of molecular genetic tools with which to manipulate peripheral neurons. This includes Cre driver lines to target distinct populations of neuron, and Cre reporter lines that allow for the probing of function through imaging and chemogenetic ablation. These technologies have enabled the lab to understand how distinct sensations such as itch versus touch are generated, and to investigate how injury triggers reorganization of the system provoking chronic pain.
Sensory neurons surrounding a blood vessel in the skin. Credit: Barenghi
Targeting sensory neurons using ligands
Based upon their work on sensory neuron diversity, the Heppenstall group has been developing pharmacological approaches with which to gain control of sensory neuron activityand inhibit pain or itch at its source. At the core of the technology, the group has engineered protein ligands which bind selectively to subtypes of neuron that provoke itch, mechanical hypersensitivity or inflammatory pain. Using these ligands they have been able to direct photosensitizers to neurons in the skin and achieve long-term inhibition of itch and pain. The lab is now exploring how these ligands can be used to deliver small molecules, proteins or genes into neurons, thus allowing for precise control over neuronal function.
Gene delivery in sensory neurons. Credit: Barenghi
Tubulin acetylation and touch
Acetylated microtubules play a critical role in regulating the sensitivity of sensory neurons to mechanical touch. The protein Atat1 is the enzyme responsible for acetylation of α-tubulin, and in its absence mice, flies and nematode worms display a profound loss of mechanical sensitivity but no other overt phenotype. The Heppenstall group generated the first mouse knockout of Atat1, and demonstrated that mice lacking α-tubulin acetylation display a profound loss of mechanical touch and pain. They are now exploiting this finding to develop small molecule inhibitors for tubulin acetylation as a novel class of analgesic
Acetylated microtubules in sensory neurons. Credit: Castaldi
Imaging sensory neurons
The Heppenstall group has a long-standing interest in developing methods for live imaging of neurons in the skin. They have focused on chemogenetic approaches that utilize genetic targeting to localize synthetic indicators to cells of interest. For example, the lab produced the first Cre dependent reporter mouse line expressing the SNAP-tag, and demonstrated that it allowed for bothmanipulation of behaviour and monitoring of cellular fluorescence in complex tissue. More recently, they have generated a semisynthetic tethered voltage indicator called STeVI1 that can be genetically targeted to neuronal membranes for optical monitoring of voltage. In ongoing work, the team are optimizing this technology to visualize how neuronal circuitry is activated by defined sensory inputs.
Snap-tag labelling of sensory neurons. Credit: Castaldi
Genetic sensory diseases
The Heppenstall laboratory has a strong interest in developing technology to treat heritable sensory diseases which cause itch and pain. They have been working on a rare skin disease called familial primary localized cutaneous amyloidosis (FPLCA) that provokes severe itching and damage to the skin. The group generated a mouse model of FPLCA and found that by targeting a photosensitizer to itch sensing neurons they were able to reverse symptoms of the disease for several months. They are now exploring whether disease causing mutations in FPLCA and other genetic diseases can be corrected in the skin through cell-directed gene editing.
Amyloid deposits in the skin of an FPLCA mouse. Credit: Nocchi