Axonal neuropathy-associated TRPV4 regulates neurotrophic factor-derived axonal growth

Abstract

Spinal muscular atrophy and hereditary motor and sensory neuropathies are characterized by muscle weakness and atrophy caused by the degenerations of peripheral motor and sensory nerves. Recent advances in genetics have resulted in the identification of missense mutations in TRPV4 in patients with these hereditary neuropathies. Neurodegeneration caused by Ca(2+) overload due to the gain-of-function mutation of TRPV4 was suggested as the molecular mechanism for the neuropathies. Despite the importance of TRPV4 mutations in causing neuropathies, the precise role of TRPV4 in the sensory/motor neurons is unknown. Here, we report that TRPV4 mediates neurotrophic factor-derived neuritogenesis in developing peripheral neurons. TRPV4 was found to be highly expressed in sensory and spinal motor neurons in early development as well as in the adult, and the overexpression or chemical activation of TRPV4 was found to promote neuritogenesis in sensory neurons as well as PC12 cells, whereas its knockdown and pharmacologic inhibition had the opposite effect. More importantly, nerve growth factor or cAMP treatment up-regulated the expression of phospholipase A(2) and TRPV4. Neurotrophic factor-derived neuritogenesis appears to be regulated by the phospholipase A(2)-mediated TRPV4 pathway. These findings show that TRPV4 mediates neurotrophic factor-induced neuritogenesis in developing peripheral nerves. Because neurotrophic factors are essential for the maintenance of peripheral nerves, these findings suggest that aberrant TRPV4 activity may lead to some types of pathology of sensory and motor nerves.

FIGURE 1.

TRPV4 was expressed in early developing peripheral neurons. A, immunostaining of neonatal mouse (P1) DRG neurons with anti-TRPV4, showing TRPV4 enrichment in sensory neurons. Scale bar, 100 μm. B, immunostaining of adult mouse (P1) DRG neurons with anti-TRPV4, showing TRPV4 enrichment in soma and nerve fibers. Scale bar, 25 μm. C and D, adult mouse DRG neurons were double stained with anti-TRPV4, neurofilament M (NF-M, a marker of myelinated neurons), and TRPV1 (a marker of nociceptors). TRPV4 was found to be expressed in both small and large neurons. Scale bar, 25 μm. E, immunostaining of adult mouse spinal cord with anti-TRPV4 and NeuN. Immunofluorescence for TRPV4 was enhanced in large motor neurons in the ventral horn (a) and in small interneurons in the dorsal horn of the spinal cord (b). F, immunostaining of an adult mouse spinal cord with anti-TRPV4 and GFAP (a glial cell marker). TRPV4 immunoreactivity was not found to colocalize with that of GFAP in the ventral (a) and dorsal horn (b) of the spinal cord. G, immunostaining of an adult mouse gastrocnemius muscles with anti-TRPV4 and α-bungarotoxin (α-BTX), a neuromuscular junction marker. α-Bungarotoxin was conjugated with Alexa Fluor® 555.

FIGURE 2.

TRPV4 transcripts increased neurite outgrowth. A, NGF induced-neuritogenesis in PC12 cells and primary cultures of DRG neurons for 6 days. To better visualize neurites (green), cells were stained with anti-TuJ1 conjugated with Alexa Fluor® 488 goat anti-mouse IgG. Scale bar, 10 μm. B, increase of TRPV4 transcript levels in NGF-treated PC12 cells. mRNA levels were quantified by RT-PCR. Data were normalized versus GAPDH. C, increases in TRPV4 transcript levels in primary cultured DRG neurons. mRNA levels were quantified by RT-PCR. Data were normalized versus GAPDH.

FIGURE 3.

Effects of TRPV4 siRNA and TRPV4 overexpression on neurite outgrowth. A, TRPV4 mRNA levels were measured by RT-PCR in control and RNAi-treated groups in PC12 cells. GAPDH was used as a positive control. B and C, TRPV4 siRNA-transfected cells showed less neurite outgrowth and fewer cells with neurites than scrambled siRNA-treated PC12 cells. Scale bar, 10 μm. D, when treated with 5,6-EET, cells expressing TRPV4 (TRPV4^WT) showed an immediate increase in [Ca²⁺]_i. In contrast, a loss of function mutant of TRPV4 (TRPV4^P19S) evoked a markedly lower Ca²⁺ response to 5,6-EET. E, the overexpression of TRPV4^WT promoted neurite growth and increased the number of cells with neurites.

FIGURE 4.

Modulation of neuritogenesis with agonists or antagonists of TRPV4. A, neurite outgrowth was observed when PC12 cells were treated with 4α-PDD or AA, but was rarely observed in cells treated with ruthenium red (RR) (a pan-TRP channel blocker). PC12 cells were immunostained with anti-Tuj1 conjugated with Alexa Fluor 488 goat anti-mouse IgG (green), and cultured for 6 days with NGF. B-D, treatment with 4α-PDD, AA, or 5,6-EET (all TRPV4 activators) significantly increased neurite lengths (B) and the percentages of PC12 cells bearing neurites (C). In contrast, 14,15-EET (an inactive metabolite of cytochrome P450 in terms of activating TRPV4) failed to increase the neurite lengths of PC12 cells or DRG neurons in primary culture (D). Scale bar, 10 μm. E, treatment of DRG neurons with GSK1016790A, a selective activator of TRPV4 significantly augmented, whereas treatment with HC-067047, a selective antagonist reduced neuritogenesis.

FIGURE 5.

The NGF-induced MAPK pathway regulated TRPV4. A, NGF (100 ng/ml) increased the levels of PLA₂ and TRPV4 transcripts in PC12 cells, and these increases were inhibited by PD098059 (a MEK inhibitor). GAPDH mRNA was use to normalize PLA₂ and TRPV4 mRNA levels. B, NGF (100 ng/ml) increased the protein expression of PLA₂ and TRPV4 in PC12 cells probed by Western blot. α-Tubulin was blotted as a control. C, inhibitors of PLA₂ (AACOCF₃) and MEK (PD098059) suppressed NGF-induced neurite growth in PC12 cells, whereas an activator of PLA₂ (melittin) promoted NGF-induced neurite growth.

FIGURE 6.

TRPV4 regulated cAMP-dependent neurite growth. A, treatment of PC12 cells with 1 mm cAMP increased the transcript levels of PLA₂ and TRPV4, but co-treatment with PD098059 inhibited these transcript level increases. B, treatment of 1 mm cAMP increased the protein expression of PLA₂ and TRPV4. C and D, PC12 cells treated with 1 mm cAMP showed markedly enhanced neurite growth, and co-treatment with 250 nm melittin augmented this growth, whereas co-treatment with 10 μm PD098095 or 20 μm AACOCF₃ suppressed cAMP-induced neurite growth (C). The TRPV4 activators, AA, 5,6-EET, and 4α-PDD augmented cAMP-induced neurite growth (D). ***, p < 0.001 versus untreated cells (CTL); ###, p < 0.001 versus cAMP-treated cells (cAMP). E and F, treatment of PC12 cells with 10 μm forskolin (FSK) significantly promoted neurite growth, and co-treatment with melittin augmented this growth, but co-treatment with PD098095 or AACOCF₃ suppressed FSK-induced neurite growth (E). The TRPV4 activators, AA, 5,6-EET, and 4α-PDD augmented cAMP-induced neurite growth (F).

FIGURE 7.

TRPV4 activation increased actin polymerization and cell adhesion. A, fluorescent photomicrograph of phalloidin, a marker for F-actin. In the presence of NGF, treatment with 10 μm 4α-PDD or 300 nm 5,6-EET induced morphological change and increased phalloidin fluorescence intensities. Phalloidin was conjugated with Alexa Fluor 488. Nuclei were stained with Hoechst 33342. Scale bar, 10 μm. NGF and TRPV4 activators were applied to cells for 24 h before fixation for staining. B, after shaking, numbers of attached cells were markedly greater after treating cells with 4α-PDD or 5,6-EET. Cell numbers were determined by staining nuclei with Hoechst 33342. C, quantification of phalloidin fluorescence intensities. 4α-PDD and 5,6-EET increased the phalloidin fluorescence intensities of cells treated with or without NGF. D, summary of the effect of treatment with TRPV4 agonists (4α-PDD or 5,6-EET) on numbers of attached cells. Note that TRPV4 activators significantly increased the number of cells attached to plates after shaking.