Peripheral mechanisms of peripheral neuropathic pain

Peripheral neuropathic pain (PNP), neuropathic pain that arises from a damage or disease affecting the peripheral nervous system, is associated with an extremely large disease burden, and there is an increasing and urgent need for new therapies for treating this disorder. In this review we have highlighted therapeutic targets that may be translated into disease modifying therapies for PNP associated with peripheral neuropathy. We have also discussed how genetic studies and novel technologies, such as optogenetics, chemogenetics and single-cell RNA-sequencing, have been increasingly successful in revealing novel mechanisms underlying PNP. Additionally, consideration of the role of non-neuronal cells and communication between the skin and sensory afferents is presented to highlight the potential use of drug treatment that could be applied topically, bypassing drug side effects. We conclude by discussing the current difficulties to the development of effective new therapies and, most importantly, how we might improve the translation of targets for peripheral neuropathic pain identified from studies in animal models to the clinic

Misregulation of mitochondria-lysosome contact dynamics in Charcot-Marie-Tooth Type 2B disease Rab7 mutant sensory peripheral neurons

Inter-organelle contact sites between mitochondria and lysosomes mediate the crosstalk and bidirectional regulation of their dynamics in health and disease. However, mitochondria-lysosome contact sites and their misregulation have not been investigated in peripheral sensory neurons. Charcot-Marie-Tooth type 2B disease is an autosomal dominant axonal neuropathy affecting peripheral sensory neurons caused by mutations in the GTPase Rab7. Using live super-resolution and confocal time-lapse microscopy, we showed that mitochondria-lysosome contact sites dynamically form in the soma and axons of peripheral sensory neurons. Interestingly, Charcot-Marie-Tooth type 2B mutant Rab7 led to prolonged mitochondria-lysosome contact site tethering preferentially in the axons of peripheral sensory neurons, due to impaired Rab7 GTP hydrolysis-mediated contact site untethering. We further generated a Charcot-Marie-Tooth type 2B mutant Rab7 knock-in mouse model which exhibited prolonged axonal mitochondria-lysosome contact site tethering and defective downstream axonal mitochondrial dynamics due to impaired Rab7 GTP hydrolysis as well as fragmented mitochondria in the axon of the sciatic nerve. Importantly, mutant Rab7 mice further demonstrated preferential sensory behavioral abnormalities and neuropathy, highlighting an important role for mutant Rab7 in driving degeneration of peripheral sensory neurons. Together, this study identifies an important role for mitochondria-lysosome contact sites in the pathogenesis of peripheral neuropathy

Visualization of Sensory Neurons and Their Projections in an Upper Motor Neuron Reporter Line.

Visualization of peripheral nervous system axons and cell bodies is important to understand their development, target recognition, and integration into complex circuitries. Numerous studies have used protein gene product (PGP) 9.5 [a.k.a. ubiquitin carboxy-terminal hydrolase L1 (UCHL1)] expression as a marker to label sensory neurons and their axons. Enhanced green fluorescent protein (eGFP) expression, under the control of UCHL1 promoter, is stable and long lasting in the UCHL1-eGFP reporter line. In addition to the genetic labeling of corticospinal motor neurons in the motor cortex and degeneration-resistant spinal motor neurons in the spinal cord, here we report that neurons of the peripheral nervous system are also fluorescently labeled in the UCHL1-eGFP reporter line. eGFP expression is turned on at embryonic ages and lasts through adulthood, allowing detailed studies of cell bodies, axons and target innervation patterns of all sensory neurons in vivo. In addition, visualization of both the sensory and the motor neurons in the same animal offers many advantages. In this report, we used UCHL1-eGFP reporter line in two different disease paradigms: diabetes and motor neuron disease. eGFP expression in sensory axons helped determine changes in epidermal nerve fiber density in a high-fat diet induced diabetes model. Our findings corroborate previous studies, and suggest that more than five months is required for significant skin denervation. Crossing UCHL1-eGFP with hSOD1G93A mice generated hSOD1G93A-UeGFP reporter line of amyotrophic lateral sclerosis, and revealed sensory nervous system defects, especially towards disease end-stage. Our studies not only emphasize the complexity of the disease in ALS, but also reveal that UCHL1-eGFP reporter line would be a valuable tool to visualize and study various aspects of sensory nervous system development and degeneration in the context of numerous diseases

UCHL1-eGFP reporter line allows visualization of peripheral nervous system <i>in vivo</i>.

<p>(A) Enhanced green fluorescent protein (eGFP) expression is evident at embryonic day (E) 12 in UCHL1-eGFP mice. (B-D) Cross-section through the E12 embryo showing eGFP⁺ trigeminal ganglia (TG; C) and dorsal root ganglia (DRG; D) neurons. (E) Cross-section of the postnatal day (P) 0 trunk reveals eGFP⁺ DRG neurons, their axons, sympathetic chain ganglia (SCG), and spinal motor neurons (SMN) in the ventral horn of the spinal cord. (F) eGFP expression in the adult DRG. Insert enlarged to the right. (G) Cross section of DRG section reveals eGFP+ neurons. (H) Sagittal-section of the P0 head showing eGFP⁺ TG neurons and their axons projecting to the whisker pad and brainstem. (I) Cross section of adult TG. (J) eGFP⁺ SCG in the exposed adult thoracic cavity. Insert enlarged to the right. (K) Open-book prep of wholemount intestines displays a network of eGFP⁺ enteric nervous system (ENS) neurons and their axons. Insert enlarged to the right. (L-M) Cross-sections of intestines show ENS neurons in both the myenteric plexus (MP; L) and submucosal plexus (SP; marked with eGFP expression; M). (N-O) eGFP is expressed in the testis. (P) eGFP is not expressed in the retina. WP: whisker pad, ONL: outer nuclear layer, INL: inner nuclear layer, GCL: ganglion cell layer. Scale bars A, B, F inset and J inset 1 mm; C 500 μm; D, E, K, N 200 μm; F, H, J 2 mm; G, I, K inset, L, M, O 50 μm; O inset 10 μm; and P 100 μm.</p

eGFP labels projections of sensory neurons in the periphery.

<p>(A-D) eGFP⁺ dorsal root ganglia (DRG) axons can be seen extending into the developing limb bud (A), in the depilated leg of the whole adult mouse (B), in the hairy back skin of the whole adult mouse both in section (C) and wholemount (D). Insert enlarged to the right. (E-G) UCHL1-eGFP reporter mouse can be used to study innervation of hair follicles in whisker pads of P0 (E-F) or adult mice (G). Insert enlarged to the right. (H) Proprioceptive nerve endings in muscle spindles are eGFP⁺. (I-K) eGFP⁺ axons can be used to study innervation of the P0 foot (I), or adult footpad epidermis and sweat glands (J, K). Insert enlarged to the right. Scale bars A, D 1 mm; A inset, B, C, E, F, G, I 500 μm; C inset, E inset, F inset, I inset 100 μm; D inset, J, K 200 μm; H 50 μm; J inset, K inset 25 μm.</p

All DRG neurons are eGFP+ and nociceptive neurons display bright GFP.

<p>(A-B) eGFP is expressed in all DRG neurons in UCHL1-eGFP reporter mice (A) and is completely absent in the wild type (WT) mice (B). (C) Pie-chart graph showing distribution of eGFP⁺ neurons. (D) eGFP co-localizes with UCHL1 in P30 DRG neurons. (E) Co-localization of bright eGFP⁺ with Parvalbumin (PV), a marker for proprioceptive neurons. (F) Bar graph representation of average percentage of bright and dim eGFP⁺ DRG neurons among PV⁺ neurons. (G) Co-localization of bright eGFP⁺ with Isolectin IB₄, a marker for nociceptive neurons. (H) Bar graph representation of average percentage of bright and dim eGFP⁺ DRG neurons among IB₄⁺ neurons. (I) Co-localization of bright eGFP⁺ with calcitonin gene related peptide (CGRP), a marker for nociceptive neurons. (J) Bar graph representation of average percentage of bright and dim eGFP⁺ DRG neurons among CGRP⁺ neurons. Bar graphs represent mean ± SEM. Student’s <i>t</i>-test, * <i>P</i> < 0.05, ** <i>P</i> < 0.01, *** <i>P</i> < 0.001. Scale bars A-B,D,E,G,I 100 μm.</p