No abstract

Recent evidence suggests that alteration of axon initial segment (AIS) geometry (i.e., length or location along the axon) contributes to CNS dysfunction in neurological diseases. For example, AIS length is shorter in the prefrontal cortex of type 2 diabetic mice with cognitive impairment.To determine the key type 2 diabetes-related factor that produces AIS shortening we modified levels of insulin, glucose, or the reactive glucose metabolite methylglyoxal in cultures of dissociated cortices from male and female mice and quantified AIS geometry using immunofluorescent imaging of the AIS proteins AnkyrinG and βIV spectrin. Neither insulin nor glucose modification altered AIS length.Exposure to 100 but not 1 or 10 µM methylglyoxal for 24 h resulted in accumulation of the methylglyoxal-derived advanced glycation end-product hydroimidazolone and produced reversible AIS shortening without cell death. Methylglyoxal-evoked AIS shortening occurred in both excitatory and putative inhibitory neuron populations and in the presence of TTX. In single-cell recordings resting membrane potential was depolarized at 0.5-3 h and returned to normal at 24 h. In multi-electrode array recordings methylglyoxal produced an immediate ~300% increase in spiking and bursting rates that returned to normal within 2 min, followed by a ~20% reduction of network activity at 0.5-3 h and restoration of activity to baseline levels at 24 h. AIS length was unchanged at 0.5-3 h despite the presence of depolarization and network activity reduction. Nevertheless, these results suggest that methylglyoxal could be a key mediator of AIS shortening and disruptor of neuronal function during type 2 diabetes. Significance StatementSmall changes in the structure of the axon initial segment affect neuronal function and may be a key mediator of neurological complications in various disease states. However, the specific disease factors that mediate structural changes at the axon initial segment are relatively unknown. This is the first study to show that increase of methylglyoxal is sufficient to reduce axon initial segment length and modulate neuronal function at the cellular and network levels. Methylglyoxal is a disease factor implicated in a wide variety of conditions including type 2 diabetes, Alzheimer's disease, and aging. Thus, these findings could significantly impact the understanding of neurological complications in several disease states and are of broad pathophysiological relevance.

Use of the immunosuppressants calcineurin inhibitors (CNIs) are limited by irreversible kidney damage, hallmarked by renal fibrosis. CNIs directly damage many renal cell types. Given the diverse renal cell populations, additional targeted cell types and signaling mechanisms warrant further investigation. We hypothesize that fibroblasts contribute to CNI-induced renal fibrosis and propagate profibrotic effects via the TGFβ/Smad signaling axis. To test this, kidney-damage resistant mice (C57BL/6) received tacrolimus (10mg/kg) or vehicle for 21 days. Renal damage markers and signaling mediators were assessed. To investigate their role in renal damage, mouse renal fibroblasts were exposed to tacrolimus (1nM) or vehicle for 24 hrs. Morphological and functional changes in addition to downstream signaling events were assessed. Tacrolimus-treated kidneys displayed evidence of renal fibrosis. Moreover, α-SMA expression was significantly increased, suggesting the presence of fibroblast activation. TGFβ receptor activation and downstream Smad2/3 signaling was also upregulated. Consistent with in vivo findings, tacrolimus-treated renal fibroblasts displayed a phenotypic switch known as fibroblast-to-myofibroblast transition (FMT), as α-SMA, actin stress fibers, cell motility and Col IV expression were significantly increased. These findings were accompanied by concomitant induction of TGFβ signaling. Pharmacological inhibition of Smad2/3 activation attenuated tacrolimus-induced phenotypic changes. These findings suggest that 1) tacrolimus inhibits calcineurin (Cn) activity/NFAT activation while inducing TGFβ ligand secretion and receptor activation in the renal fibroblast, 2) aberrant TGFβ receptor activation stimulates Smad-mediated production of myofibroblast markers, promoting FMT and 3) FMT contributes to extracellular matrix (ECM) expansion in tacrolimus-induced renal fibrosis. These results incorporate renal fibroblasts to the growing list of CNI-targeted cell types and establish renal FMT as a process mediated via a TGFβ-dependent mechanism.