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. 2019 Nov 22:21:720-735.
doi: 10.1016/j.isci.2019.10.064. Epub 2019 Oct 31.

Single-Cell RNA-seq Reveals Profound Alterations in Mechanosensitive Dorsal Root Ganglion Neurons with Vitamin E Deficiency

Carrie J Finno  1 , Janel Peterson  2 , Mincheol Kang  3 , Seojin Park  3 , Matthew H Bordbari  2 , Blythe Durbin-Johnson  4 , Matthew Settles  4 , Maria C Perez-Flores  3 , Jeong H Lee  3 , Ebenezer N Yamoah  3
Affiliations

Single-Cell RNA-seq Reveals Profound Alterations in Mechanosensitive Dorsal Root Ganglion Neurons with Vitamin E Deficiency

Carrie J Finno et al. iScience. .

Abstract

Ninety percent of Americans consume less than the estimated average requirements of dietary vitamin E (vitE). Severe vitE deficiency due to genetic mutations in the tocopherol transfer protein (TTPA) in humans results in ataxia with vitE deficiency (AVED), with proprioceptive deficits and somatosensory degeneration arising from dorsal root ganglia neurons (DRGNs). Single-cell RNA-sequencing of DRGNs was performed in Ttpa-/- mice, an established model of AVED. In stark contrast to expected changes in proprioceptive neurons, Ttpa-/- DRGNs showed marked upregulation of voltage-gated Ca2+ and K+ channels in mechanosensitive, tyrosine-hydroxylase positive (TH+) DRGNs. The ensuing significant conductance changes resulted in reduced excitability in mechanosensitive Ttpa-/- DRGNs. A highly supplemented vitE diet (600 mg dl-α-tocopheryl acetate/kg diet) prevented the cellular and molecular alterations and improved mechanosensation. VitE deficiency profoundly alters the molecular signature and functional properties of mechanosensitive TH+ DRGN, representing an intriguing shift of the prevailing paradigm from proprioception to mechanical sensation.

Keywords: Molecular Neuroscience; Neuroscience; Transcriptomics.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
An Overall Increase in the Number of Differentially Expressed Transcripts across All DRGN Subpopulations with α-TOH Deficiency (A) A t-distributed stochastic neighbor embedding (t-SNE) plot of merged datasets of the six experimental mice to define neuronal subpopulations clusters. Thirteen clusters were identified based on previously reported gene expression profiles (Li et al., 2016, Usoskin et al., 2015). The “NP2-2” subgroup contained some genes representative of the NP2 cluster but was distinct from the NP2 cluster and has been previously characterized (Usoskin et al., 2015, Li et al., 2016). (B) An increasing number of significantly (PFDR<0.05) differentially expressed transcripts (DETs) was identified in each DRGN subpopulation with increasing contrasts of vitE deficiency (i.e. SUPP vs. DEF > WT vs. DEF > SUPP vs. WT). (C and D) (C) The two most commonly dysregulated transcripts across DRGN subpopulations were carbonic anhydrase 8 (Car8), which was significantly downregulated in 10/13 DRG clusters with vitE deficiency (SUPP vs. DEF), and (D) the lineage-specific transcription factor Runx3, which was significantly upregulated in 10/13 DRG clusters with vitE deficiency (SUPP vs. DEF). p values were adjusted by a false discovery rate of 0.05 and log-transformed. Significance was set at PFDR<0.05, corresponding to a –log Padjusted>1.3 (red line). NF = neurofilament, NP = non-peptidergic, PEP = peptidergic, TH = tyrosine hydroxylase, UNKNOWN = unknown cluster, n = 2 mice per group with ~3,600 cells/mouse profiled.
Figure 2
Figure 2
Upregulation Intermediate Voltage-Gated Ca2+ and K+ Channels in TH+ DRGNs with vitE Deficiency Heatmaps, plotted by –logPadjusted, comparing the degree of upregulation for R-type intermediate voltage-gated Ca2+, Cacna1e, and Ca2+-activated K+ channel beta subunits channels, Kcnmb1 and Kcnmb2, in DRGN subpopulations with vitE deficiency. Contrast A = SUPP vs. WT, contrast B = WT vs. DEF, contrast C = SUPP vs DEF. Cacna1e= Cav2.3 intermediate voltage-activated Ca2+ channel, Kcnmb = Potassium large conductance calcium-activated channel, subfamily M, beta.
Figure 3
Figure 3
Increase in Cacna1e and Kcnmb2 with vitE Deficiency in TH+ DRG (A–D) Green: Th, Blue: DAPI nuclei, Red: Cacna1e [(A) mRNA, (B) protein] and Kcnmb2 [(C) mRNA, (D) protein]. White box inset magnified in the last column. Fluorescent immunohistochemistry from 4-month WT, DEF, and SUPP mice, n = 1–2 per group. Scale bars represent 10 μm (TH, Cacna1e, Kcnbm2, and merge) and 3 μm (enlarged). (E) Quantification of mRNA using RNAscope for Cacna1e and Kcnmb2. Mean ± SD, N = 8 counts per experimental group, one-way ANOVA, or Kruskal-Wallis. Scale bars represent 10 μm (TH, Cacna1e, Kcnbm2, and merge) and 3 μm (enlarged). (F) Von Frey assay, demonstrating a significant increase in sensitivity (i.e. lower Dixon's score) in the SUPP vs. DEF mice. Mean ± SD, N = 8–22 per group, one-way ANOVA, ****p< 0.0001, **p< 0.01, *p< 0.05.
Figure 4
Figure 4
Membrane Properties of Small-Diameter Dorsal Root Ganglion Neurons (DRGNs) from WT and DEF Mice Current-clamp recordings were performed on DRGNs 6-month-old mice. Membrane input resistance (Ri) was determined by evaluating membrane voltage changes in response to negative and positive current injection. The ohmic relations were fitted with linear regression and the Ri derived from the slope. (A) Representative traces of displacement-clamp currents recorded using CsCl/NMG-based pipette solution in response to ~250-ms mechanical displacement steps of ~0.42 μm to WT small-diameter DRGN (shown as inset). DRGNs were held at −70 mV. Summary data of displacement-response relationship of mechanically activated (MA) currents (IMA) represented as the I/Imax or open channel probability (Po) against displacement (X) fitted with single Boltzmann function. Data from WT DRGNs (shown in black symbols and fitted with sigmoidal curve in black) and the one-half maximum displacements (X1/2) are 1.1 ± 0.1 μm and 0.3 ± 0.1 μm (n = 11). Data from DEF DRGNs (shown in blue symbols and fitted with sigmoidal curve in blue) and the one-half maximum displacements (X1/2) are 1.5 ± 0.1 μm and 0.4 ± 0.1 μm (n = 7). (B) Among the small-diameter neurons, there were three distinct classes: fast, medium, and slow adapting. Exemplary plots from fast-adapting DRGNs from WT mice (shown in black, mean Ri in MΩ; 100 ± 6; n = 15), and in DEF (shown in blue, mean Ri in MΩ; 45 ± 5; n = 17: p< 0.0001). The inset is an example of data used to generate the plots. For medium-adapting neurons the Ri (in MW) were as follows: WT (306 ± 23; n = 9) and DEF (162 ± 29; n = 11: p = 0.0014). For slow-adapting neurons the Ri were WT (626 ± 47; n = 13) and DEF (395 ± 38; n = 11: p = 0.0012). (C) Brief (~5 ms) stepwise positive current was injected to elicit subthreshold (shown in different color codes) and threshold depolarization (WT in black and DEF in blue). The threshold currents are indicated. The threshold voltage was determined, using a dV/dt loop plot (inset, right). (D and E) Typical voltage response from slow-adapting DRGNs recorded from WT and DEF mice. (F and G) Action potentials generated using varying pulse durations from fast-adapting DRGNs in WT (F) and DEF (G) mice. (H) Plots of the relations between threshold potential and pulse duration in WT (in black) and DEF (in blue) mice. The insets show the dV/dt versus membrane potential (V) loops used to determine the thresholds.
Figure 5
Figure 5
Increased K+ and Ca2+ Current Density in DEF versus WT DRGNs (A)Whole-cell onward K+ currents were elicited using depolarizing steps from −110 to 40 mV (ΔV = 10 mV). The tail currents were at −40 mV. Current traces recorded from WT and DEF DRGNs are shown in black and blue, respectively. To obtain a profile of currents that are enhanced in DEF DRGNs, we determined the “difference currents” between DEF and WT neurons at −70 and 40 mV step voltages (traces are plotted with dashed lines in inset). (B) Summary of steady-state currents was normalized to individual membrane capacitance (Cm), from 6-month-old WT mice (shown with black line and symbol) and DEF (shown with gray traces). Data were generated from 14 DRGNs from each experimental group. The mean current densities (in pA/pF) in WT and DEF DRGNs at 0 mV step voltage were 26.1 ± 2.6 and 39.9 ± 2.8; n = 14, p = 0.0015. (C and D) Inward Ca2+ currents recorded from a 12-pF DRGNs in WT (in black) and DEF (in blue) mice from −90 and −40 mV holding potentials. Currents were generated using voltage steps ranging from −110 to 40 mV. The difference-current traces (−90 mV) - (−40 mV) are plotted in dashed lines as an inset. (E) Peak Ca+ current density (I)-voltage (V) relation from data amassed from 12 DRGNs in each group. The current densities generated from a holding voltage of −40 mV are plotted with WT in black and DEF in blue. The high-voltage activated component of the Ca2+ current was enhanced in the DEF DRGNs. The peak current density (in pA/pF) for currents elicited from a holding potential of −40 mV for WT DRGNs was 20.1 ± 1.5 (n = 9) and for DEF DRGNs was 30.9 ± 2.5 (n = 9, p = 0.002). After application of 500 nM rSNX-482 to the DEF DRGNs the peak current density plummeted to 12.6 ± 1.2 (n = 6, p< 0.0001).
Figure 6
Figure 6
Membrane Properties of Small-Diameter Dorsal Root Ganglion Neurons (DRGNs) from SUPP Mice (A) Current-clamp recordings were performed on DRGNs 6-month-old mice supplemented with 600 mg dL-alpha-tocopheryl/kg feed. Data were assessed from DRGNs with capacitance <15 pF. Membrane input resistance (Ri) was determined by evaluating membrane voltage changes in response to negative and positive current injection. (B) The ohmic relations were fitted with linear regression and the Ri derived from the slope. SUPP mice fast-adapting DRGNs had a mean Ri of 115 ± 9 MW (n = 11). (C) Similar to WT DRGNs, as the pulse duration was prolonged, the threshold voltage declined. (D) Typical voltage response from slow-adapting DRGNs recorded from SUPP mice. Compared with WT DRGNs, the slow-adapting neurons in SUPP did not recover fully despite vitE supplementation. (E)Whole-cell onward K+ currents were elicited using depolarizing steps from −130 to 30 mV (ΔV = 10 mV), from a holding potential of −90 mV. The tail currents were at −60 mV. Current traces recorded from SUPP DRGNs are shown. (F) Summary of steady-state currents was normalized to individual membrane capacitance (Cm), from 6-month-old SUPP mice. Data were generated from 15 DRGNs. (G and H) Inward Ca2+ currents recorded from a 10-pF DRGNs in SUPP mice from −90 and −40 mV holding potentials. Currents were generated using voltage steps ranging from −120 to 40 mV. (I) The current-voltage relations generated at −90 mV and −40 mV holding potential is plotted. The I-V relations of Ca2+ currents from DEF DRGNs from Figure 5E is re-plotted in blue for comparison.
Figure 7
Figure 7
Proposed Mechanism of Action for α-TOH in TH+ DRGN With adequate α-TOH (left), constitutive activity of the RAR-related orphan receptor alpha (RORA) transcription factor is maintained, increasing IP3R1 transcription (Gold et al., 2003, Sarachana and Hu, 2013). Although there is evidence that vitE can affect the plasma membrane structure and bind to signaling enzymes to affect their activity (Zingg, 2015, Habermehl et al., 2005), we propose in this model that signaling through the PLC/IP3/IP3R1 axis maintains Ca2+ homeostasis. VitE can suppress PLC activity (Domijan et al., 2014) and, by stimulating DAGK (Koya et al., 1997), DAG is removed and PKC inhibited, providing a protective effect. With α-TOH deficiency (right), cholesterol is oxidized and resulting oxysterols repress constitutive RORA activity (Wang et al., 2010), leading to decreased IP3R1 transcription. ROS activate the PLC/IP3/IP3R1 axis (Servitja et al., 2000, Vaarmann et al., 2010); however, without sufficient IP3R1, [Ca2+]i cannot increase. Additionally, loss of DAGK stimulation increases DAG and PKC. We propose this leads to the identified alterations in membrane excitability and activation of apoptotic pathways in DRGNs. BK= big potassium channel; DAG = diaglycerol; DAGK= diaglycerol kinase; IP3 = inositol triphosphate; IP3R1 = inositol 1,4,5 triphosphate receptor 1; PIP2 = phosphatidylinositol 4,5-bisphosphate; PKC = protein kinase C; PLC = phospholipase C; RORA= RAR-related orphan receptor alpha; ROS = reactive oxygen species.
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