Identifying the proteins of the cochlear mechanoelectrical transduction machinery that are also involved in the development of auditory cortex interneurons.

Project Information

DEVINAC

Grant agreement ID: 793417

DOI

10.3030/793417

Project closed

EC signature date 27 March 2018

Start date 1 July 2019

End date 4 November 2021

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 173 076,00

EU contribution

€ 173 076,00

Coordinated by

INSTITUT PASTEUR
France

Periodic Reporting for period 1 - DEVINAC (Identifying the proteins of the cochlear mechanoelectrical transduction machinery that are also involved in the development of auditory cortex interneurons.)

Reporting period: 2019-07-01 to 2021-06-30

Summary of the context and overall objectives of the project

Hearing loss is a major concern and serious burden for Public Health as it is affecting 466 million worldwide according to the World Health Organization. Genetic research into hereditary forms of deafness in humans has largely contributed to the deciphering of the molecular physiology of the auditory sensory organ, the cochlea. Since the discovery of the first gene responsible for deafness in both humans and mice, the Usher syndrome gene encoding myosin-VIIa, about 110 genes responsible for non-syndromic forms of deafness and about 300 genes responsible for syndromic forms have been reported in humans and/or mice. By contrast, the genetic approach has provided little information about the central auditory system. One possible explanation for this discrepancy is that intrinsic auditory central dysfunction may be concealed by peripheral deficits in some genetic forms of deafness. Indeed, my hosting team recently discovered that the Usher syndrome genes encoding protocadherin-15 (cdhr15) and cadherin 23 (cdhr23), the cadherin-related proteins forming the tip links are also expressed by precursors of a subpopulation of GABAergic inhibitory parvalbumin interneurons in the auditory cortex. The extent to which central deficits are masked by peripheral ones in genetic forms of deafness is currently unknown. This evaluation is of particular importance because early prosthetic intervention is based on the revival of auditory cortex microcircuits, setting new challenges for clinical practice and the development of adapted auditory rehabilitation methods.

The team I integrated is interested in addressing possible central auditory deficits in genetic forms of deafness. Our previous results demonstrating the coexistence of intrinsic central auditory deficits with peripheral deficits point to the importance of extending this study to other molecules. To date, eight other proteins make up the mechanoelectrical transduction molecular machinery in the hair bundle: through their interaction with cdhr23 and cdhr15, they form the upper and the lower tip-link complex, respectively. Therefore, the possibility that the entire Usher syndrome 1 complex and other molecular components of the mechanoelectrical transduction machinery are involved in the development of cdhr15/cdhr23-expressing auditory cortex interneuron precursors is an attractive working hypothesis.
Taking this into account, my project was divided in two main aims:
Aim 1) To determine which proteins of the mechanoelectrical transduction machinery, in addition to cdhr15 and cdhr23, are involved in the development of cortical interneurons.
Aim 2) To determine whether cdhr15 and cdhr23 are essential for the migration of interneuron precursors to the cortex and for their integration within auditory cortex microcircuits.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

My hosting team identified and characterized the role of 3 Usher proteins in the auditory cortex. They demonstrated that many GABAergic interneurons require for their normal development two cadherin-related (cdhr) proteins, cdhr15 and cdhr23, that form the hair bundle tip links gating the mechanoelectrical transduction channels. Mutant mice lacking either protein showed a major decrease in the number of some inhibitory neurons the auditory cortex, and displayed audiogenic reflex seizures. In addition, in the absence of adhesion G protein-coupled receptor V1 (adgrv1), we observed a similar decrease of inhibitory neurons in the auditory cortex. These results were published in PNAS in July 2017 (Libé-Philippot et al., PNAS. 2017 114(30):7765-7774.).

To continue on this work, during my Marie Curie action I designed and produced new genetic tools that express cre recombinase under the control of the Cdhr23 and Cdhr15 promoters i) to characterize the neuronal populations expressing Cdhr15 or Cdhr23 by fluorescent tracing in the auditory cortex and in the brain, and ii) to manipulate the activity of Cdhr15/Cdhr23-expressing cells. Results from Cdhr23-Cre knock-in mice suggest that several populations of cells throughout the brain express Cdhr23. In addition, we observed many projecting neurons in the auditory cortex that originate from Cdhr23-expressing neurons.

In parallel, I implemented the RNAscope technique in the lab, allowing the detection of mRNA expression for target genes in the brain. Using this technique, I obtained additional results on the role of Cdhr15 showing that Cdhr15 was expressed in some inhibitory neurons and also in a subtype of glial cells involved in the stabilization of cortical interneurons through their myelination.

In parallel, I identified an additional Usher protein in the auditory cortex. This protein is a mechanoelectrical transduction complex protein for which cre knock-in mice was already available. Through RNAscope and high-throughput single-cell transcriptomics, we could confirm that cells expressing this protein in the temporal cortex are excitatory neurons. The analysis of these brains were performed using a new software, NeuroInfo, allowing the mapping of neuroanatomical structures in histological sections. Using this software, I characterized the localization of cells that were expressing this MET complex protein during their development and could show that they are mainly located in different layers of the cortex, with a higher density of cells in layer 4.

The data from the two main objectives of my project are currently being used to write two manuscripts that are in preparation for a future submission process. These data have also been presented in a national and in an international meeting. Some of the data have been used in a presentation to general public.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Hearing impairment is the most frequent sensory defect. It affects over 6% of the world’s population, with around 50%–60% having genetic aetiology. The vast majority of these genetic forms of deafness involve defects in the auditory sensory organ, the cochlea. In addition to peripheral deficits, these genetic forms of deafness may also affect the central auditory system. Indeed, complete or partial auditory deprivation of peripheral origin has indirect deleterious effects for the maturation of the central auditory system, including the auditory cortex. Moreover, increasing number of proteins encoded by causal genes of peripheral deafness are being found to have direct or intrinsic roles in the central auditory system. Such associated central intrinsic deficits would probably be masked by the peripheral deficits.

There is growing perception that central auditory deficits must be taken into account in the management of genetic forms of peripheral deafness. My work on characterizing cortical deficits in mouse models of preciously identified forms of peripheral human deafness is of particular relevance on understanding the extent to which patients with hereditary forms of deafness also have from cortical deficiencies. Thus, most genetic forms of deafness should no longer be considered as isolated deficits of the cochlea. This evaluation is of particular importance because early prosthetic intervention is based on the revival of auditory cortex microcircuits. Our results should lead to an improvement of hearing rehabilitation strategies in patients and pave the way for the development of a genetic approach for studying the cellular and molecular mechanisms involved in auditory cortex development and functioning.

Usher protein in the adult mouse brain (tdTomato, red)