Axonal Regrowth of Olfactory Sensory Neurons In Vitro.

One of the most prevalent causes of olfactory loss includes traumatic brain injury with subsequent shearing of olfactory axons at the level of the cribriform plate (anterior skull base). Scar tissue at this level may prevent axonal regrowth toward the olfactory bulb. Currently, there is no cure for this debilitating and often permanent condition. One promising therapeutic concept is to implant a synthetic scaffold with growth factors through the cribriform plate/scar tissue to induce neuroregeneration. The first step toward this goal is to investigate the optimum conditions (growth factors, extracellular matrix proteins) to boost this regeneration. However, the lack of a specifically tailored in vitro model and an automated procedure for quantifying axonal length limits our ability to address this issue. The aim of this study is to create an automated quantification tool to measure axonal length and to determine the ideal growth factors and extracellular proteins to enhance axonal regrowth of olfactory sensory neurons in a mouse organotypic 2D model. We harvested olfactory epithelium (OE) of C57BL/6 mice and cultured them during 15 days on coverslips coated with various extracellular matrix proteins (Fibronectin, Collagen IV, Laminin, none) and different growth factors: fibroblast growth factor 2 (FGF2), brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), nerve growth factor (NGF), retinoic acid (RA), transforming growth factor ß (TGFß), and none. We measured the attachment rate on coverslips, the presence of cellular and axonal outgrowth, and finally, the total axonal length with a newly developed automated high-throughput quantification tool. Whereas the coatings did not influence attachment and neuronal outgrowth rates, the total axonal length was enhanced on fibronectin and collagen IV (p = 0.001). The optimum growth factor supplementation media to culture OE compared to the control condition were as follows: FGF2 alone and FGF2 from day 0 to 7 followed by FGF2 in combination with NGF from day 7 to 15 (p < 0.0001). The automated quantification tool to measure axonal length outperformed the standard Neuron J application by reducing the average analysis time from 22 to 3 min per specimen. In conclusion, robust regeneration of murine olfactory neurons in vitro can be induced, controlled, and efficiently measured using an automated quantification tool. These results will help advance the therapeutic concept closer toward preclinical studies.

Inhibitory Kcnip2 neurons of the spinal dorsal horn control behavioral sensitivity to environmental cold.

Proper sensing of ambient temperature is of utmost importance for the survival of euthermic animals, including humans. While considerable progress has been made in our understanding of temperature sensors and transduction mechanisms, the higher-order neural circuits processing such information are still only incompletely understood. Using intersectional genetics in combination with circuit tracing and functional neuron manipulation, we identified Kcnip2-expressing inhibitory (Kcnip2GlyT2) interneurons of the mouse spinal dorsal horn as critical elements of a neural circuit that tunes sensitivity to cold. Diphtheria toxin-mediated ablation of these neurons increased cold sensitivity without affecting responses to other somatosensory modalities, while their chemogenetic activation reduced cold and also heat sensitivity. We also show that Kcnip2GlyT2 neurons become activated preferentially upon exposure to cold temperatures and subsequently inhibit spinal nociceptive output neurons that project to the lateral parabrachial nucleus. Our results thus identify a hitherto unknown spinal circuit that tunes cold sensitivity.

WNT Activation and TGFß-Smad Inhibition Potentiate Stemness of Mammalian Auditory Neuroprogenitors for High-Throughput Generation of Functional Auditory Neurons In Vitro.

Hearing loss affects over 460 million people worldwide and is a major socioeconomic burden. Both genetic and environmental factors (i.e., noise overexposure, ototoxic drug treatment and ageing), promote the irreversible degeneration of cochlear hair cells and associated auditory neurons, leading to sensorineural hearing loss. In contrast to birds, fish and amphibians, the mammalian inner ear is virtually unable to regenerate due to the limited stemness of auditory progenitors, and no causal treatment is able to prevent or reverse hearing loss. As of today, a main limitation for the development of otoprotective or otoregenerative therapies is the lack of efficient preclinical models compatible with high-throughput screening of drug candidates. Currently, the research field mainly relies on primary organotypic inner ear cultures, resulting in high variability, low throughput, high associated costs and ethical concerns. We previously identified and characterized the phoenix auditory neuroprogenitors (ANPGs) as highly proliferative progenitor cells isolated from the A/J mouse cochlea. In the present study, we aim at identifying the signaling pathways responsible for the intrinsic high stemness of phoenix ANPGs. A transcriptomic comparison of traditionally low-stemness ANPGs, isolated from C57Bl/6 and A/J mice at early passages, and high-stemness phoenix ANPGs was performed, allowing the identification of several differentially expressed pathways. Based on differentially regulated pathways, we developed a reprogramming protocol to induce high stemness in presenescent ANPGs (i.e., from C57Bl6 mouse). The pharmacological combination of the WNT agonist (CHIR99021) and TGFß/Smad inhibitors (LDN193189 and SB431542) resulted in a dramatic increase in presenescent neurosphere growth, and the possibility to expand ANPGs is virtually limitless. As with the phoenix ANPGs, stemness-induced ANPGs could be frozen and thawed, enabling distribution to other laboratories. Importantly, even after 20 passages, stemness-induced ANPGs retained their ability to differentiate into electrophysiologically mature type I auditory neurons. Both stemness-induced and phoenix ANPGs resolve a main bottleneck in the field, allowing efficient, high-throughput, low-cost and 3R-compatible in vitro screening of otoprotective and otoregenerative drug candidates. This study may also add new perspectives to the field of inner ear regeneration.

Intrinsically Self-renewing Neuroprogenitors From the A/J Mouse Spiral Ganglion as Virtually Unlimited Source of Mature Auditory Neurons.

Nearly 460 million individuals are affected by sensorineural hearing loss (SNHL), one of the most common human sensory disorders. In mammals, hearing loss is permanent due to the lack of efficient regenerative capacity of the sensory epithelia and spiral ganglion neurons (SGN). Sphere-forming progenitor cells can be isolated from the mammalian inner ear and give rise to inner ear specific cell types in vitro. However, the self-renewing capacities of auditory progenitor cells from the sensory and neuronal compartment are limited to few passages, even after adding powerful growth factor cocktails. Here, we provide phenotypical and functional characterization of a new pool of auditory progenitors as sustainable source for sphere-derived auditory neurons. The so-called phoenix auditory neuroprogenitors, isolated from the A/J mouse spiral ganglion, exhibit robust intrinsic self-renewal properties beyond 40 passages. At any passage or freezing-thawing cycle, phoenix spheres can be efficiently differentiated into mature spiral ganglion cells by withdrawing growth factors. The differentiated cells express both neuronal and glial cell phenotypic markers and exhibit similar functional properties as mouse spiral ganglion primary explants and human sphere-derived spiral ganglion cells. In contrast to other rodent models aiming at sustained production of auditory neurons, no genetic transformation of the progenitors is needed. Phoenix spheres therefore represent an interesting starting point to further investigate self-renewal in the mammalian inner ear, which is still far from any clinical application. In the meantime, phoenix spheres already offer an unlimited source of mammalian auditory neurons for high-throughput screens while substantially reducing the numbers of animals needed.

Olfactory dysfunction in frontotemporal dementia and psychiatric disorders: A systematic review.

Frontotemporal dementia (FTD) is a progressive neurodegenerative disease. Diagnosis of FTD, especially the behavioural variant, is challenging because of symptomatic overlap with psychiatric disorders (depression, schizophrenia, bipolar disorder). Olfactory dysfunction is common in both FTD and psychiatric disorders, and often appears years before symptom onset. This systematic review analysed 74 studies on olfactory function in FTD, depression, schizophrenia and bipolar disorder to identify differences in olfactory dysfunction profiles, focusing on the most common smell measures: odour identification and discrimination. Results revealed that FTD patients were severely impaired in odour identification but not discrimination; in contrast, patients diagnosed with schizophrenia showed impairments in both measures, while those diagnosed with depression showed no olfactory impairments. Findings in bipolar disorder were mixed. Therefore, testing odour identification and discrimination differentiates FTD from depression and schizophrenia, but not from bipolar disorder. Given the high prevalence of odour identification impairments in FTD, and that smell dysfunction predicts neurodegeneration in other diseases, olfactory testing seems a promising avenue towards improving diagnosis between FTD and psychiatric disorders.

Olfactory Fluctuation Revisited.

OBJECTIVES: Many patients complain about olfactory fluctuation (OF), which is a symptom commonly attributed to sinonasal disease. Data-based evidence for its association with sinonasal disease is scarce. The aim of the study is to identify explanatory variables associated with OF and to analyze its predictive value regarding sinonasal disease. STUDY DESIGN: We performed a retrospective study based on patients with olfactory dysfunction. METHODS: We analyzed data from 482 patients attending the smell and taste outpatient clinic with full psychophysical workup and structured questions regarding their symptoms. The questionnaire included items on OF and chronic nasal symptoms. Clinical investigators filled out the second part of this questionnaire that included information about nasal endoscopy, psychophysical tests of orthonasal olfaction (Sniffin' Sticks), retronasal olfaction, and putative etiology of olfactory dysfunction. RESULTS: OF was more prevalent in sinonasal disease (42.4%) compared to other putative etiologies of olfactory dysfunction such as postinfectious (28%) or posttraumatic (11.7%) (X2 [5, n = 440] = 24.98; P < .0001). OF was strongly associated with Sniffin' Sticks score categories (anosmia, hyposmia, normosmia) (X2 [2, n = 424] = 39.21; P < .0001; Cramer's V = 0.30; P < .0001) and presence of "chronic nasal symptoms" (X2 [1, n = 437] = 22.71; P < .0001; Cramer's V = 0.23; P < .0001). The accuracy in predicting putative sinonasal disease etiology when OF was present depended strongly on the clinical context. CONCLUSION: Olfactory fluctuation is a symptom mostly but not exclusively associated with sinonasal disease, elevated Sniffin' Sticks test scores, and is frequently accompanied by other nasal complaints. Its presence is valuable information for clinicians to be integrated into the clinical context when doing patients' workup. LEVEL OF EVIDENCE: 4 Laryngoscope, 130:2442-2447, 2020.