An exPADItion for citrullination in the developing hair follicle

During epidermal development, to assure proper tissue structure, highly complex transcriptional networks interact within the stem cell compartments of the epidermis and hair follicles (HFs) to balance the choice between self-renewal or differentiation. The full characterisation of the protein profiles resulting from those transcriptional networks, within the compartments of the HF, remains, however, incomplete. Moreover, the proteins themselves can be regulated via posttranslational modification (PTMs). One such PTM is citrullination, carried out by the peptidylarginine deiminase (PADI) family of enzymes. Although, PADIs have been described in other stem and progenitor cells, their role in hair follicle stem cell (HFSC) and progenitor lineages have remained elusive. The main objectives of this thesis are to address the functional consequences of PADI expression in HFSCs during development. Paper I identifies Padi4 expression in the developing HF, where it is found to participate in restricting proliferation and lineage commitment of HF progenitors, as well as playing a role in the central mechanism for translational control, and by doing so altering the distinct sequential events that mark HF differentiation progression. As a result, we identify citrullination as a means to assert regulation of protein function in HFSCs and progenitors. Paper II identifies alternative isoforms of PADI2 and PADI3, in oligodendrocytes and HF differentiated cells, respectively, and show that the alternative isoforms have an incumbering effect on the enzymatic activity and stability of their conventional counterparts. Paper III is a review paper in which meta-analysis of published human citrullinomes in health and inflammatory disease reveals that citrullination is a commonplace yet highly dynamic molecular regulator of protein function. A strong case is made for the involvement of PADIs and citrullination in hair follicle stem cell biology and inflammatory alopecia. Paper IV addresses the involvement of transcription factor ID1 in self-renewal and differentiation of epidermal progenitor cells during development. This study describes how ID1 facilitates synchronisation of progenitor proliferation and differentiation via TCF3- binding, and establishes a novel axis of coordination for how BMP-induction of Id1 expression via pSMAD1/5 is supressed by CEBPa. The combined efforts within this thesis demonstrate the clear and overarching importance of PADIs and citrullination in skin developmental physiology

GLAST Deficiency in Mice Exacerbates Gap Detection Deficits in a Model of Salicylate-Induced Tinnitus

Gap detection or gap pre-pulse inhibition of the acoustic startle (GPIAS) has been successfully used in rat and guinea pig models of tinnitus, yet this system has proven to have low efficacy in CBA mice, with low basal GPIAS and subtle tinnitus like effects. Here, we tested five mouse strains (CBA, BalbC, CD-1, C57BL/6 and sv129) for pre-pulse inhibition and gap detection with varying interstimulus intervals (ISI) and found the that mice from a CBA genetic background had the poorest capacities of suppressing the startle response in presence of a pre-pulse or a gap. CD-1 mice displayed variable responses throughout all ISI. Interestingly, C57BL/6, sv129 and BalbC showed efficient suppression with either pre-pulses or gaps with shorter ISI. The glutamate aspartate transporter (GLAST) is expressed in support cells from the cochlea and buffers the excess of glutamate. We hypothesized that loss of GLAST function could sensitize the ear to tinnitus-inducing agents, such as salicylate. Using shorter ISI to obtain a greater dynamic range to assess tinnitus-like effects, we found that disruption of gap detection by salicylate was exacerbated across various intensities of a 32 kHz narrow band noise gap carrier in GLAST KO mice when compared to their wild-type littermates. Auditory brainstem responses (ABR) and distortion products of otoacoustic emission (DPOAE) were performed to evaluate the effects on hearing functions. Salicylate caused greater auditory threshold shifts (near 15 dB) in GLAST KO mice than in wild-type mice across all tested frequencies, despite similarly reduced DPOAE. Despite these changes, inhibition using broad-band gap carriers and 32 kHz pre-pulses were not affected. Our study suggests that GLAST deficiency could become a useful experimental model to decipher the mechanisms underlying drug-induced tinnitus. Future studies addressing the neurological correlates of tinnitus in this model could provide additional insights into the mechanisms of tinnitus

Effect of N-acetylcysteine infusion on exercise-induced modulation of insulin sensitivity and signaling pathways in human skeletal muscle

Reactive oxygen species (ROS) produced in skeletal muscle may play a role in potentiating the beneficial responses to exercise; however, the effects of exercise-induced ROS on insulin action and protein signaling in humans has not been fully elucidated. Seven healthy, recreationally active participants volunteered for this double-blind, randomized, repeated-measures crossover study. Exercise was undertaken with infusion of saline (CON) or the antioxidant N-acetylcysteine (NAC) to attenuate ROS. Participants performed two 1-h cycling exercise sessions 7–14 days apart, 55 min at 65% V̇o2peak plus 5 min at 85%V̇o2peak, followed 3 h later by a 2-h hyperinsulinemic euglycemic clamp (40 mIU·min−1·m2) to determine insulin sensitivity. Four muscle biopsies were taken on each trial day, at baseline before NAC infusion (BASE), after exercise (EX), after 3-h recovery (REC), and post-insulin clamp (PI). Exercise, ROS, and insulin action on protein phosphorylation were evaluated with immunoblotting. NAC tended to decrease postexercise markers of the ROS/protein carbonylation ratio by −13.5% (P = 0.08) and increase the GSH/GSSG ratio twofold vs. CON (P < 0.05). Insulin sensitivity was reduced (−5.9%, P < 0.05) by NAC compared with CON without decreased phosphorylation of Akt or AS160. Whereas p-mTOR was not significantly decreased by NAC after EX or REC, phosphorylation of the downstream protein synthesis target kinase p70S6K was blunted by 48% at PI with NAC compared with CON (P < 0.05). We conclude that NAC infusion attenuated muscle ROS and postexercise insulin sensitivity independent of Akt signaling. ROS also played a role in normal p70S6K phosphorylation in response to insulin stimulation in human skeletal muscle