Tackling Hearing Loss with Synthetic Chemistry
By Christine Hamadani
Hearing loss is a major public health concern in the US, associated with difficulty hearing and perceiving sound in absence of an external stimulus. To first understand how hearing works, we need to look within the inner ear, where there’s a little spiral-shaped organ encased in bone. This organ is the cochlea, whose job is to pick up sound information via sensory hair cells, translate it, and send the information to brain via nerve cells called spiral ganglion neurons (SGNs). Age-related hearing loss has classically been associated with loss of two types of cells: cochlear sensory hair cells (HCs) and first-order auditory spiral ganglion neurons (SGNs). An additional, “hidden” etiology of hearing loss is the loss of synaptic connections between cochlear sensory hair cells and first-order cochlear neurons. Detecting this loss ranges from difficult to impossible when using conventional hearing testing methods. It is therefore of great interest to promote survival of spiral ganglion neurons and to regenerate synapses between SGNS and hair cells after synaptic loss following exposure to chemicals that are toxic to the ear, or noise damage.
As such, we at the Jung lab at the Eaton Peabody Laboratories, Harvard Medical School and MEEI, collaborating with the University of Southern California, have developed a novel small molecule approach for targeted delivery of a protein called a neurotrophin to the cochlea to reconnect spiral ganglion neurons (SGN) and hair cells. Neurotrophins are proteins that promote neuronal outgrowth and wiring.
A small neurotrophin molecule, known as 7, 8-dihydroxyflavone (DHF), specifically activates the tropomyosin receptor kinase B (TrkB) pathway that promotes neuronal survival and stimulates the growth of neural projections, known as neurite outgrowth. It has been shown to have similar activity to brain-derived neurotrophic factor (BDNF), one of two primary neurotrophins expressed in the cochlea, but with its small size, maintains higher ability to travel across the blood-brain barrier, a biological filter that only lets specific substances in from the bloodstream to the brain. Bisphosphonates are molecules that are widely used to increase bone strength and density by binding to the surfaces of bones. The Jung lab has previously demonstrated in a guinea pig model that a fluorescently labeled bisphosphonate, when administered to the round window membrane (a membrane allowing substances to cross from the middle to inner ear), penetrated the membrane and diffused throughout the cochlea. As they have an affinity for cochlear bone, bisphosphonates may be used for targeted delivery of neurotrophic agents to the SGNs to promote long-term survival, neurite outgrowth, and the regeneration of synapses between IHCs and SGNs.
To create the novel bisphosphonate coupled to 7,8-DHF (known as Ris-DHF) with organic chemistry, a carboxyl group (black) was first added to the para position of 7, 8-Dihydroxyflavone (DHF)’s phenyl ring (blue), seen in the schematic below:
Hearing loss is a major public health concern in the US, associated with difficulty hearing and perceiving sound in absence of an external stimulus. To first understand how hearing works, we need to look within the inner ear, where there’s a little spiral-shaped organ encased in bone. This organ is the cochlea, whose job is to pick up sound information via sensory hair cells, translate it, and send the information to brain via nerve cells called spiral ganglion neurons (SGNs). Age-related hearing loss has classically been associated with loss of two types of cells: cochlear sensory hair cells (HCs) and first-order auditory spiral ganglion neurons (SGNs). An additional, “hidden” etiology of hearing loss is the loss of synaptic connections between cochlear sensory hair cells and first-order cochlear neurons. Detecting this loss ranges from difficult to impossible when using conventional hearing testing methods. It is therefore of great interest to promote survival of spiral ganglion neurons and to regenerate synapses between SGNS and hair cells after synaptic loss following exposure to chemicals that are toxic to the ear, or noise damage.
As such, we at the Jung lab at the Eaton Peabody Laboratories, Harvard Medical School and MEEI, collaborating with the University of Southern California, have developed a novel small molecule approach for targeted delivery of a protein called a neurotrophin to the cochlea to reconnect spiral ganglion neurons (SGN) and hair cells. Neurotrophins are proteins that promote neuronal outgrowth and wiring.
A small neurotrophin molecule, known as 7, 8-dihydroxyflavone (DHF), specifically activates the tropomyosin receptor kinase B (TrkB) pathway that promotes neuronal survival and stimulates the growth of neural projections, known as neurite outgrowth. It has been shown to have similar activity to brain-derived neurotrophic factor (BDNF), one of two primary neurotrophins expressed in the cochlea, but with its small size, maintains higher ability to travel across the blood-brain barrier, a biological filter that only lets specific substances in from the bloodstream to the brain. Bisphosphonates are molecules that are widely used to increase bone strength and density by binding to the surfaces of bones. The Jung lab has previously demonstrated in a guinea pig model that a fluorescently labeled bisphosphonate, when administered to the round window membrane (a membrane allowing substances to cross from the middle to inner ear), penetrated the membrane and diffused throughout the cochlea. As they have an affinity for cochlear bone, bisphosphonates may be used for targeted delivery of neurotrophic agents to the SGNs to promote long-term survival, neurite outgrowth, and the regeneration of synapses between IHCs and SGNs.
To create the novel bisphosphonate coupled to 7,8-DHF (known as Ris-DHF) with organic chemistry, a carboxyl group (black) was first added to the para position of 7, 8-Dihydroxyflavone (DHF)’s phenyl ring (blue), seen in the schematic below:
This generated a DHF intermediate product, available to react with the bisphosphonate Risedronate to form hybrid molecule Ris-DHF. Afterward, to measure Ris-DHF’s effect on neurite outgrowth, SGNs were dissected in vitro from postnatal mouse cochleae and treated with Risedronate, DHF, Ris-DHF hybrid, or plain media control. To assess Ris-DHF’s ability to stimulate synaptic regeneration, dissected organ of Corti (OC) cultured with attached SGNs were treated with Kainic Acid (KA), thought to mimic excessive neural stimulation and kill ribbon synapses, which are the sites of contact near the sensory hair cells from which critical signals are produced to relay sound information. Following KA treatment, explants were treated with Risedronate, Ris-DHF, or DHF. To evaluate Ris-DHF’s functional ability after first binding it to bone particles in vitro (to be able to simulate its behavior in vivo within the bony inner ear), the SGN explant assay protocol was performed after first treating hydroxyapatite (the mineral form of calcium apatite, which makes up tooth bone) with Risedronate, Ris-DHF, and DHF.
Notably, it was found that Ris-DHF stimulated a significantly higher relative ratio of neurite outgrowth than untreated control and Risedronate alone. Moreover, Ris-DHF, like DHF, promoted significant synaptic regeneration in the KA explant assay. Finally, Ris-DHF drove the highest levels of neurite outgrowth after pre-binding to hydroxyapatite pellets. Thus, Ris-DHF increases neurite outgrowth in vitro and maintains this ability after binding to hydroxyapatite. In addition, Ris-DHF regenerates synapses in vitro following synaptic loss.
Very recent auditory brainstem measurements show that when it is delivered to the round window membrane in vivo post-noise exposure, Ris-DHF is able to functionally restore hearing relative to non-exposed wild-type age-matched mice. Thus, these molecules may have promise as a novel approach to targeted delivery of drugs to treat hearing loss. These findings have incredibly large clinical implications, potentially offering a non-invasive long-term solution to restore hearing for a diverse group of patients, ranging from rock concert fans to war veterans and seniors, who currently have limited treatment options such as hearing aids or largely invasive cochlear implant surgery. By getting the drug to exactly where it needs to go, our solution can treat the problem right at the source and may improve the quality of life for millions.
