Mitochondrial dysfunction in an animal model of diabetic neuropathy is associated with a reduction of neurosteroid synthesis.

Introduction

Diabetic neuropathy is a common cause of painful neuropathy, and treatment is often suboptimal because the underlying aetiology is poorly understood. Peripheral and central sensitisation are implicated in the development of neuropathic pain with neuroplasticity occurring at multiple levels of the pain pathway (Harvey & Dickenson, 2008). GABAergic neurones at all levels of the pain pathway have a vital role in the transmission of painful stimuli in the perception of pain itself (D’Mello & Dickenson, 2008). Endogenous and exogenous neurosteroids may act as potent positive allosteric modulators of GABA_A receptors (GABA_ARs) and consequently exhibit analgesic, anxiolytic, anticonvulsant, and sedative properties (D'Hulst et al., 2009).

Within inhibitory synapses, the presynaptic fusion of a single vesicle releases the inhibitory neurotransmitter GABA to activate synaptic GABA_ARs. Under voltage-clamp conditions this causes a miniature inhibitory postsynaptic current (mIPSC). Drugs that enhance GABA_AR function cause a prolongation of the mIPSC decay phase. Recent work in a model of type-II diabetic neuropathy (ob/ob) revealed that layer 2/3 cortical pyramidal neurones of the pain pathway exhibited reduced endogenous neurosteroid modulation of the GABA_AR, and exogenously applied neurosteroids had an exaggerated impact (Humble, 2016a). The mechanism responsible appeared unrelated to GABA_AR sensitivity, but instead was associated with a reduction of neurosteroid precursors, such as pregnenolone, which is metabolised sequentially to the active compound allopregnanolone (Figure 1) (Humble, 2013; Humble, 2016a). Pregnenolone is synthesised in the mitochondrion from its precursor cholesterol by the side chain cleavage enzyme P450 located in the inner mitochondrial membrane (Do Rego et al., 2009; Schumacher et al., 2012), and it is postulated that diabetic neuropathy may be associated with a reduction in mitochondrial activity. Cholesterol is translocated across the mitochondrial membrane by the 18 kDa translocator protein (TSPO) in a coordinated fashion with the steroidogenic acute regulatory (StAR) protein (Do Rego et al., 2009; Gatliff & Campanella, 2016; Rupprecht et al., 2010; Stocco et al., 2017; Figure 1).

Figure 1. Modulation of the GABA_AR by endogenous neurosteroids.

Cholesterol is taken through the mitochondrial membrane by the translocator protein (TSPO) where it is converted to pregnenolone by the cytochrome P450 side chain cleavage enzyme. Pregnenolone is converted to progesterone by 3β-hydroxysteroid dehydrogenase (3β-HSD), which is in turn reduced to dihydroxyprogesterone by 5α-reductase (5α-R). Dihydroxyprogesterone is converted to allopregnanolone by 3α-hydroxysteroid dehydrogenase (3α-HSD). Postsynaptic GABA_ARs are activated by GABA that has been released from vesicles in the presynaptic nerve terminal. GABA induces a conformational change of the GABA_AR, opening its central channel and thereby allowing the passage of chloride ions and the subsequent generation of miniature inhibitory postsynaptic currents (mIPSCs). The negative chloride ions induce hyperpolarisation of the neuronal membrane, which mediates neuronal inhibition. Neurosteroids, such as the active compound allopregnanolone, modulate GABA_AR function and facilitate inhibition of the neuronal membrane. (Humble, 2013). The translocation of cholesterol into the mitochondria by TSPO is the first rate-limiting step and is enhanced by the presence of specific ligands such as diazepam. Thus diazepam may enhance GABA_AR modulation by binding to the GABA_AR directly and separately by increased neurosteroidogenesis. This modulation may be selectively inhibited at the GABA_AR itself by the antagonist flumazenil and separately by the 5α-R inhibitor finasteride, which inhibits neurosteroidogenisis.

The present study explored the impact of the benzodiazepine diazepam, a positive allosteric modulator of the GABA_AR (D’Hulst et al., 2009), on GABA_AR modulation via neurosteroidogenesis in diabetic and wild type (WT) mice. Benzodiazepines are also known to activate neurosteroidogenesis by binding to TSPO (Rupprecht et al., 2010; Tokuda et al., 2010).

Methods

The methods are identical to those published by the same author previously (Humble, 2016a), with the exception of the drugs diazepam and flumazenil, which were not used in the previous study. Diazepam and flumazenil were purchased (Tocris, Bristol UK) and prepared as concentrated stock solutions in dimethyl sulfoxide before being added to the artificial extracellular solution as per the previous study (Humble, 2016a).

Results

Prolonged exposure (2 hrs) of mature cortical neurones to diazepam (1μM) in the presence of flumazenil had an exaggerated effect on the cortical GABA_AR-mediated mIPSCs of ob/ob mice in comparison to WT mice

Whole-cell voltage-clamp recordings were made in L2/3 cortical neurones of WT and ob/ob mice after at least two hours of incubation with diazepam, flumazenil and finasteride. Diazepam alone had the same effect on both strains of mice. In the WT mice, flumazenil inhibited the effect of diazepam (τ_W: control = 4.0 ± 0.1 ms, n = 35; finasteride 50 μM = 4.2 ± 0.1 ms, n = 7; diazepam 1 μM = 5.9 ± 0.2 ms, n = 6; flumazenil 10 μM & diazepam 1 μM = 4.0 ± 0.2 ms, n = 7; flumazenil 10 μM, finasteride 50 μM & diazepam 1 μM = 4.0 ± 0.3 ms, n = 6; One-way ANOVA, P <0.05; post hoc Newman Keul’s test revealed a difference only for diazepam 1 μM, P <0.05; Figure 2). By contrast, in the ob/ob mice, flumazenil only partially inhibited the effect of diazepam, and the persisting effect of diazepam in the presence of flumazenil in the ob/ob mice could be prevented by the presence of the 5α-reductase enzyme inhibitor finasteride (τ_W: ob/ob control = 3.5 ± 0.1 ms, n = 25; finasteride 50 μM = 3.7 ± 0.2 ms, n = 6; diazepam 1 μM = 5.7 ± 0.3 ms, n = 6; flumazenil 10 μM & diazepam 1 μM = 4.9 ± 0.3 ms, n = 6; flumazenil 10 μM, finasteride 50 μM & diazepam 1 μM = 3.7 ± 0.1 ms, n = 5; One-way ANOVA, P <0.05; post hoc Newman Keul’s test revealed significant intergroup differences for the flumazenil groups, P <0.05; Figure 2).

Figure 2. Prolonged exposure (2 hrs) of mature cortical neurones to diazepam (1 μM) in the presence of flumazenil had an exaggerated effect on the cortical GABA_AR-mediated mIPSCs of ob/ob mice in comparison to WT mice.

(A) Superimposed exemplar averaged GABA_AR-mediated miniature inhibitory postsynaptic currents (mIPSCs) acquired from a representative WT cortical neurone and from equivalent neurones after ~2 hours pre-incubation of the brain slice with diazepam (1 μM), flumazenil (10 μM) and finasteride (50 μM). (B) Superimposed exemplar averaged GABA_AR-mediated mIPSCs acquired from a representative ob/ob cortical neurone and from equivalent neurones after ~2 hours pre-incubation of the brain slice with diazepam (1μM), flumazenil (10 μM) and finasteride (50 μM). (C) Histogram illustrating that flumazenil is able to prevent the effect of diazepam to prolong the duration of the GABA_AR-mediated mIPSC in WT cortical neurones, but only has a partial efficacy in ob/ob cortical neurones (τ_w in ms; one-way ANOVA, P >0.05; Post hoc Newman Keul’s test). The persisting effect of diazepam in the presence of flumazenil in the ob/ob mice could be prevented by the presence of the 5α-reductase enzyme inhibitor finasteride (τ_w in ms; one-way ANOVA, P >0.05; Post hoc Newman Keul’s test). Ctrl = control; Finast = finasteride; Diaz = diazepam; Flumaz = flumazenil.

Discussion

Layer 2/3 cortical neurones from mature type-II diabetic ob/ob are known to have a reduced endogenous pregnane-derived neurosteroid tone in comparison to strain matched WT controls (Humble, 2016a). The present data indicate that by promoting the uptake of pregnenolone’s precursor cholesterol by the mitochondria, via TSPO, diazepam may rescue the reduced neurosteroid tone. The restored neurosteroid tone could re-establish GABA_AR-mediated neuro-inhibitory tone in cases of neuropathic hypersensitivity. With specific reference to these data, the key result is the difference in response of the WT and ob/ob to simultaneous incubation with diazepam and flumazenil. In contrast, diazepam and the 5α-reductase inhibitor finasteride individually or in combination produced the same response in both WT and ob/ob. This may be interpreted as follows: in the WT, the primary effect of diazepam incubation is direct allosteric modulation of the GABA_AR, with negligible contribution from neurosteroidogenesis via mitochondrial TSPO activation. In comparison, diazepam has an exaggerated effect on GABAergic inhibitory tone in the ob/ob, despite the presence of the GABA_AR benzodiazepine antagonist flumazenil. This effect is likely observed due to physiological upregulation of the key rate-limiting enzymes involved in neurosteroidogenesis in response to the reduced pregnenolone synthesis by the mitochondria (Figure 1; Humble, 2016a). Thus by increasing the availability of the neurosteroid precursor pregnenolone via TSPO activation, it is possible to promote enhanced neurosteroidogenesis and thereby increase GABAergic inhibitory tone via an alternate route. Benzodiazepines modulate the GABA_AR by binding to the α-γ subunit interface (D'Hulst et al., 2009), while neurosteroids bind the GABA_AR from a cavity within the α-subunit domain and modulate it directly via the α-β subunit interface (Hosie et al., 2006). There have already been a number of other studies of ligands for the mitochondrial TSPO, as this is a promising target (Gatliff & Campanella, 2016; Giatti et al., 2009; Papadopoulos & Lecanu, 2009; Rupprecht et al., 2010; Zhang et al., 2016). Considering the findings in this paper alongside previous work (Humble, 2016a) it appears that mitochondrial dysfunction may play an important role in the development of type 2 diabetic neuropathy. In this context, it follows that enhancing the GABAergic neurosteroid tone directly or indirectly could be of potential therapeutic benefit for diabetic neuropathic pain and hypersensitivity.

Data availability

Open Science Framework: Dataset of ‘Neurosteroids are reduced in diabetic neuropathy and may be associated with the development of neuropathic pain’, doi: 10.17605/osf.io/bk3tw (Humble, 2016b). Raw data for the present study can be found in Diazepam.zip.

Please refer to (Humble, 2016a) for details of standard software used for data analysis.