Abnormal calcium homeostasis in peripheral neuropathies

Abstract

Abnormal neuronal calcium (Ca2+) homeostasis has been implicated in numerous diseases of the nervous system. The pathogenesis of two increasingly common disorders of the peripheral nervous system, namely neuropathic pain and diabetic polyneuropathy, has been associated with aberrant Ca2+ channel expression and function. Here we review the current state of knowledge regarding the role of Ca2+ dyshomeostasis and associated mitochondrial dysfunction in painful and diabetic neuropathies. The central impact of both alterations of Ca2+ signalling at the plasma membrane and also intracellular Ca2+ handling on sensory neurone function is discussed and related to abnormal endoplasmic reticulum performance. We also present new data highlighting sub-optimal axonal Ca2+ signalling in diabetic neuropathy and discuss the putative role for this abnormality in the induction of axonal degeneration in peripheral neuropathies. The accumulating evidence implicating Ca2+ dysregulation in both painful and degenerative neuropathies, along with recent advances in understanding of regional variations in Ca2+ channel and pump structures, makes modulation of neuronal Ca2+ handling an increasingly viable approach for therapeutic interventions against the painful and degenerative aspects of many peripheral neuropathies.

Figure 1. Axonal Ca²⁺ signals are abnormal in neurons cultured from STZ-diabetic rats compared with age-matched control

Transient ER Ca²⁺ release and extracellular Ca²⁺ influx were induced by treatment with 20 mM caffeine or 40 mM KCl, respectively, in axons of adult lumbar DRG neurons cultured for 3 days from 4–5 month STZ-diabetic and age-matched control rats using Fluo4-AM Ca²⁺ ion detector. A: Real-time confocal images at X100 of the free Ca²⁺ ion at baseline and ER Ca²⁺ release subsequent to 1 sec of 20 mM caffeine treatment using a perfusion system. Cells were grown in Hams F12 defined medium with N2 additives (without insulin). Control neurons were treated with 10 mM D-glucose and 10 nM insulin and diabetic neurons exposed to 25 mM D-glucose and no insulin. Bar = 20 µm. B: Traces of Fluo4-AM fluorescence intensity level and bar chart of the area under the curve reflect the quantification of the ER Ca²⁺ release in response to 20 mM caffeine. Red arrow indicates point of caffeine addition. **P<0.01. C: Results with 40 mM KCl treatment. Values are means ± SEM, ***P<0.001, n=6–13 axons.

Figure 2. The mitochondrial membrane in the axons of normal neurons show greater levels of FCCP-induced depolarization compared with neurons from diabetic rats

A: Fluorescence video confocal microscopy of mitochondria in live cultures of sensory neurons of dorsal root ganglion (DRG) isolated from adult age matched or 4–5 month STZ-diabetic rats. DRG neurons were cultured in defined Hams F12 media supplemented with N2 additives (no insulin) and 10 mM D-glucose (control; with 10 nM insulin) or 25 mM D-glucose (diabetic) for 24hr. Relative levels of mitochondrial inner membrane potential were determined by staining neurons with 3 nM tetramethyl rhodamine methyl ester (TMRM; Molecular Probes, Eugene, OR) for 1 hr and detecting the fluorescence signal with a Carl Zeiss LSM510 confocal microscope. FCCP was injected into the culture media to a final concentration of 2 µM at 5 min following baseline fluorescence measurements. B: Quantification of TMRM fluorescence levels in sub-quench mode (where decreased fluorescence intensity indicates reduced mitochondrial inner membrane potential) in the axons of cultured DRG neurons. The black arrow shows FCCP injection. Values are means ± SEM, n= 65–85 axons, *P < 0.001, Students t-test.