Probing interneuronal cell communication via optogenetic stimulation

This study uses an all-optical approach to probe interneuronal communication between spiral ganglion neurons (SGNs) and neurons of other functional units, in this case cortex neurons (CNs) and hippocampus neurons (HNs), for the first time. We combined a channelrhodopsin variant (CheRiff) with a red genetically encoded calcium indicator (jRCaMP1a), enabling simultaneous optical stimulation and recording from spatially separated small neuronal populations. Stimulation of SGNs was possible with both optogenetic manipulated HNs and CNs, respectively. Furthermore, a dependency on the pulse duration of the stimulating light in regard to the evoked calcium response in the SGNs was also observed. Our results pave the way to enable innovative technologies based on “biohybrid” systems utilizing the functional interaction between different biological (eg, neural) systems. This can enable improved treatment of neurological and sensorineural disorders such as hearing loss

Successful treatment of noise-induced hearing loss by mesenchymal stromal cells: An RNAseq analysis of protective/repair pathways

Mesenchymal stromal cells (MSCs) are an adult derived stem cell-like population that has been shown to mediate repair in a wide range of degenerative disorders. The protective effects of MSCs are mainly mediated by the release of growth factors and cytokines thereby modulating the diseased environment and the immune system. Within the inner ear, MSCs have been shown protective against tissue damage induced by sound and a variety of ototoxins. To better understand the mechanism of action of MSCs in the inner ear, mice were exposed to narrow band noise. After exposure, MSCs derived from human umbilical cord Wharton\u27s jelly were injected into the perilymph. Controls consisted of mice exposed to sound trauma only. Forty-eight hours post-cell delivery, total RNA was extracted from the cochlea and RNAseq performed to evaluate the gene expression induced by the cell therapy. Changes in gene expression were grouped together based on gene ontology classification. A separate cohort of animals was treated in a similar fashion and allowed to survive for 2 weeks post-cell therapy and hearing outcomes determined. Treatment with MSCs after severe sound trauma induced a moderate hearing protective effect. MSC treatment resulted in an up-regulation of genes related to immune modulation, hypoxia response, mitochondrial function and regulation of apoptosis. There was a down-regulation of genes related to synaptic remodeling, calcium homeostasis and the extracellular matrix. Application of MSCs may provide a novel approach to treating sound trauma induced hearing loss and may aid in the identification of novel strategies to protect hearing

Development of Neuronal Guidance Fibers for Stimulating Electrodes: Basic Construction and Delivery of a Growth Factor

State-of-the-art treatment for sensorineural hearing loss is based on electrical stimulation of residual spiral ganglion neurons (SGNs) with cochlear implants (CIs). Due to the anatomical gap between the electrode contacts of the CI and the residual afferent fibers of the SGNs, spatial spreading of the stimulation signal hampers focused neuronal stimulation. Also, the efficiency of a CI is limited because SGNs degenerate over time due to loss of trophic support. A promising option to close the anatomical gap is to install fibers as artificial nerve guidance structures on the surface of the implant and install on these fibers drug delivery systems releasing neuroprotective agents. Here, we describe the first steps in this direction. In the present study, suture yarns made of biodegradable polymers (polyglycolide/poly-ε-caprolactone) serve as the basic fiber material. In addition to the unmodified fiber, also fibers modified with amine groups were employed. Cell culture investigations with NIH 3T3 fibroblasts attested good cytocompatibility to both types of fibers. The fibers were then coated with the extracellular matrix component heparan sulfate (HS) as a biomimetic of the extracellular matrix. HS is known to bind, stabilize, modulate, and sustainably release growth factors. Here, we loaded the HS-carrying fibers with the brain-derived neurotrophic factor (BDNF) which is known to act neuroprotectively. Release of this neurotrophic factor from the fibers was followed over a period of 110 days. Cell culture investigations with spiral ganglion cells, using the supernatants from the release studies, showed that the BDNF delivered from the fibers drastically increased the survival rate of SGNs in vitro. Thus, biodegradable polymer fibers with attached HS and loaded with BDNF are suitable for the protection and support of SGNs. Moreover, they present a promising base material for the further development towards a future neuronal guiding scaffold. Copyright © 2022 Wille, Harre, Oehmichen, Lindemann, Menzel, Ehlert, Lenarz, Warnecke and Behrens

Identification of a Thyroid Hormone Binding Site in Hsp90 with Implications for Its Interaction with Thyroid Hormone Receptor Beta

While many proteins are known clients of heat shock protein 90 (Hsp90), it is unclear whether the transcription factor, thyroid hormone receptor beta (TRb), interacts with Hsp90 to control hormonal perception and signaling. Higher Hsp90 expression in mouse fibroblasts was elicited by the addition of triiodothyronine (T3). T3 bound to Hsp90 and enhanced adenosine triphosphate (ATP) binding of Hsp90 due to a specific binding site for T3, as identified by molecular docking experiments. The binding of TRb to Hsp90 was prevented by T3 or by the thyroid mimetic sobetirome. Purified recombinant TRb trapped Hsp90 from cell lysate or purified Hsp90 in pull-down experiments. The affinity of Hsp90 for TRb was 124 nM. Furthermore, T3 induced the release of bound TRb from Hsp90, which was shown by streptavidin-conjugated quantum dot (SAv-QD) masking assay. The data indicate that the T3 interaction with TRb and Hsp90 may be an amplifier of the cellular stress response by blocking Hsp90 activity

Blockade of IL-36 Receptor Signaling Does Not Prevent from TNF-Induced Arthritis

Introduction: Interleukin (IL)-36a is a newly described member of the IL-1 cytokine family with a known inflammatory and pathogenic function in psoriasis. Recently, we could demonstrate that the receptor (IL-36R), its ligand IL-36a and its antagonist IL-36Ra are expressed in synovial tissue of arthritis patients. Furthermore, IL-36a induces MAP-kinase and NFkB signaling in human synovial fibroblasts with subsequent expression and secretion of pro-inflammatory cytokines. Methods: To understand the pathomechanism of IL-36 dependent inflammation, we investigated the biological impact of IL-36a signaling in the hTNFtg mouse. Also the impact on osteoclastogenesis by IL-36a was tested in murine and human osteoclast assays. Results: Diseased mice showed an increased expression of IL-36R and IL-36a in inflamed knee joints compared to wildtype controls. However, preventively treating mice with an IL-36R blocking antibody led to no changes in clinical onset and pattern of disease. Furthermore, blockade of IL-36 signaling did not change histological signs of TNF-induced arthritis. Additionally, no alteration on bone homeostasis was observed in ex vivo murine and human osteoclast differentiation assays. Conclusion: Thus we conclude that IL-36a does not affect the development of inflammatory arthritis

Identification of a Thyroid Hormone Binding Site in Hsp90 with Implications for Its Interaction with Thyroid Hormone Receptor Beta

While many proteins are known clients of heat shock protein 90 (Hsp90), it is unclear whether the transcription factor, thyroid hormone receptor beta (TRb), interacts with Hsp90 to control hormonal perception and signaling. Higher Hsp90 expression in mouse fibroblasts was elicited by the addition of triiodothyronine (T3). T3 bound to Hsp90 and enhanced adenosine triphosphate (ATP) binding of Hsp90 due to a specific binding site for T3, as identified by molecular docking experiments. The binding of TRb to Hsp90 was prevented by T3 or by the thyroid mimetic sobetirome. Purified recombinant TRb trapped Hsp90 from cell lysate or purified Hsp90 in pull-down experiments. The affinity of Hsp90 for TRb was 124 nM. Furthermore, T3 induced the release of bound TRb from Hsp90, which was shown by streptavidin-conjugated quantum dot (SAv-QD) masking assay. The data indicate that the T3 interaction with TRb and Hsp90 may be an amplifier of the cellular stress response by blocking Hsp90 activity

Unaltered clinical signs of arthritis in anti-IL36R treated hTNFtg mice.

<p>Clinical parameters: (A) body weight, (B) grip strength, (C) paw thickness and (D) joint thickness were regularly assessed in anti-IL36R treated hTNFtg mice between 4 and 8 weeks of age. Values represent the mean ±SEM (PBS control group n = 8; treatment group n = 9).</p

Increased expression of IL36R and IL36α in hTNFtg mice.

<p>otal knee RNA from knee-joints of 6-week-old male wild-type and hTNFtg mice was isolated and quantitative real-time PCR for IL-36 family members was performed. (A) IL36R, (B) IL-36α and (C) IL-36Ra. Relative Expression was calculated from the ratio of the gene of interest to the housekeeping gene β-actin (n = 7–10). Graphs depict mean ±SEM. *p≤0,05, **p≤0,01, ***p≤0,001.</p

Osteoclastogenesis is not influenced by IL-36α treatment.

<p>(A) Quantification of human osteoclast assays from four different donors, done in two independent experiments; each of them was done in triplicates, treated with rhIL36α alone, rhIL36α plus IL36RA or rhIL36α plus recombinant receptor. (B) Quantitative real-time PCR for CtsK, IL-1RAcP and IL-36R of human osteoclast precursor cells stimulated with M-CSF and RANKL to achieve osteoclast differentiation. Relative Expression was calculated from the ratio of the gene of interest to the housekeeping gene Hsp90ab1. Data from three (CtsK) or four (IL-1RAcP and IL-36R) independent donors are shown. (C) Representative pictures and quantification of three independent murine osteoclast assay untreated (control) or stimulated with 100 ng/ml recombinant murine IL36α. (D) Quantitative real-time PCR for CtsK and TRAP of murine osteoclast precursor cells stimulated with M-CSF, M-CSF and RANKL or M-CSF, RANKL and rmIL-36α to achieve osteoclast differentiation. Data from three independent experiments are shown. Relative expression was calculated from the ratio of the gene of interest to the housekeeping gene β-actin. Graphs depict mean ±SEM.</p