Tetrahydroquinoline-Capped Histone Deacetylase 6 Inhibitor SW-101 Ameliorates Pathological Phenotypes in a Charcot-Marie-Tooth Type 2A Mouse Model.

Histone deacetylase 6 (HDAC6) is a promising therapeutic target for the treatment of neurodegenerative disorders. SW-100 (1a), a phenylhydroxamate-based HDAC6 inhibitor (HDAC6i) bearing a tetrahydroquinoline (THQ) capping group, is a highly potent and selective HDAC6i that was shown to be effective in mouse models of Fragile X syndrome and Charcot-Marie-Tooth disease type 2A (CMT2A). In this study, we report the discovery of a new THQ-capped HDAC6i, termed SW-101 (1s), that possesses excellent HDAC6 potency and selectivity, together with markedly improved metabolic stability and druglike properties compared to SW-100 (1a). X-ray crystallography data reveal the molecular basis of HDAC6 inhibition by SW-101 (1s). Importantly, we demonstrate that SW-101 (1s) treatment elevates the impaired level of acetylated α-tubulin in the distal sciatic nerve, counteracts progressive motor dysfunction, and ameliorates neuropathic symptoms in a CMT2A mouse model bearing mutant MFN2. Taken together, these results bode well for the further development of SW-101 (1s) as a disease-modifying HDAC6i.

HDAC6 inhibition promotes α-tubulin acetylation and ameliorates CMT2A peripheral neuropathy in mice.

Charcot-Marie-Tooth type 2A (CMT2A) peripheral neuropathy, the most common axonal form of CMT, is caused by dominantly inherited point mutations in the Mitofusin 2 (Mfn2) gene. It is characterized by progressive length-dependent degeneration of motor and sensory nerves with corresponding clinical features of motor and sensory impairment. There is no cure for CMT, and therapeutic approaches are limited to physical therapy, orthopedic devices, surgery, and analgesics. In this study we focus on histone deacetylase 6 (HDAC6) as a therapeutic target in a mouse model of mutant MFN2 (MFN2R94Q)-induced CMT2A. We report that these mice display progressive motor and sensory dysfunction as well as a significant decrease in α-tubulin acetylation in distal segments of long peripheral nerves. Treatment with a new, highly selective HDAC6 inhibitor, SW-100, was able to restore α-tubulin acetylation and ameliorate motor and sensory dysfunction when given either prior to or after the onset of symptoms. To confirm HDAC6 is the target for ameliorating the CMT2A phenotype, we show that genetic deletion of Hdac6 in CMT2A mice prevents the development of motor and sensory dysfunction. Our findings suggest α-tubulin acetylation defects in distal parts of nerves as a pathogenic mechanism and HDAC6 as a therapeutic target for CMT2A.

Deacetylation of Miro1 by HDAC6 blocks mitochondrial transport and mediates axon growth inhibition.

Inhibition of histone deacetylase 6 (HDAC6) was shown to support axon growth on the nonpermissive substrates myelin-associated glycoprotein (MAG) and chondroitin sulfate proteoglycans (CSPGs). Though HDAC6 deacetylates α-tubulin, we find that another HDAC6 substrate contributes to this axon growth failure. HDAC6 is known to impact transport of mitochondria, and we show that mitochondria accumulate in distal axons after HDAC6 inhibition. Miro and Milton proteins link mitochondria to motor proteins for axon transport. Exposing neurons to MAG and CSPGs decreases acetylation of Miro1 on Lysine 105 (K105) and decreases axonal mitochondrial transport. HDAC6 inhibition increases acetylated Miro1 in axons, and acetyl-mimetic Miro1 K105Q prevents CSPG-dependent decreases in mitochondrial transport and axon growth. MAG- and CSPG-dependent deacetylation of Miro1 requires RhoA/ROCK activation and downstream intracellular Ca2+ increase, and Miro1 K105Q prevents the decrease in axonal mitochondria seen with activated RhoA and elevated Ca2+ These data point to HDAC6-dependent deacetylation of Miro1 as a mediator of axon growth inhibition through decreased mitochondrial transport.

α-Tubulin Acetyltransferase Is a Novel Target Mediating Neurite Growth Inhibitory Effects of Chondroitin Sulfate Proteoglycans and Myelin-Associated Glycoprotein.

Damage to the CNS results in neuronal and axonal degeneration, and subsequent neurological dysfunction. Endogenous repair in the CNS is impeded by inhibitory chemical and physical barriers, such as chondroitin sulfate proteoglycans (CSPGs) and myelin-associated glycoprotein (MAG), which prevent axon regeneration. Previously, it has been demonstrated that the inhibition of axonal histone deacetylase-6 (HDAC6) can promote microtubule α-tubulin acetylation and restore the growth of CSPGs- and MAG-inhibited axons. Since the acetylation of α-tubulin is regulated by two opposing enzymes, HDAC6 (deacetylation) and α-tubulin acetyltransferase-1 (αTAT1; acetylation), we have investigated the regulation of these enzymes downstream of a growth inhibitory signal. Our findings show that exposure of primary mouse cortical neurons to soluble CSPGs and MAG substrates cause an acute and RhoA-kinase-dependent reduction in α-tubulin acetylation and αTAT1 protein levels, without changes to either HDAC6 levels or HDAC6 activity. The CSPGs- and MAG-induced reduction in αTAT1 occurs primarily in the distal and middle regions of neurites and reconstitution of αTAT1, either by Rho-associated kinase (ROCK) inhibition or lentiviral-mediated αTAT1 overexpression, can restore neurite growth. Lastly, we demonstrate that CSPGs and MAG signaling decreases αTAT1 levels posttranscriptionally via a ROCK-dependent increase in αTAT1 protein turnover. Together, these findings define αTAT1 as a novel potential therapeutic target for ameliorating CNS injury characterized by growth inhibitory substrates that are prohibitive to axonal regeneration.

Small-molecule inhibition of STOML3 oligomerization reverses pathological mechanical hypersensitivity.

The skin is equipped with specialized mechanoreceptors that allow the perception of the slightest brush. Indeed, some mechanoreceptors can detect even nanometer-scale movements. Movement is transformed into electrical signals via the gating of mechanically activated ion channels at sensory endings in the skin. The sensitivity of Piezo mechanically gated ion channels is controlled by stomatin-like protein-3 (STOML3), which is required for normal mechanoreceptor function. Here we identify small-molecule inhibitors of STOML3 oligomerization that reversibly reduce the sensitivity of mechanically gated currents in sensory neurons and silence mechanoreceptors in vivo. STOML3 inhibitors in the skin also reversibly attenuate fine touch perception in normal mice. Under pathophysiological conditions following nerve injury or diabetic neuropathy, the slightest touch can produce pain, and here STOML3 inhibitors can reverse mechanical hypersensitivity. Thus, small molecules applied locally to the skin can be used to modulate touch and may represent peripherally available drugs to treat tactile-driven pain following neuropathy.

TRPV1 receptor in the human trigeminal ganglion and spinal nucleus: immunohistochemical localization and comparison with the neuropeptides CGRP and SP.

This work presents new data concerning the immunohistochemical occurrence of the transient receptor potential vanilloid type-1 (TRPV1) receptor in the human trigeminal ganglion (TG) and spinal nucleus of subjects at different ontogenetic stages, from prenatal life to postnatal old age. Comparisons are made with the sensory neuropeptides calcitonin gene-related peptide (CGRP) and substance P (SP). TRPV1-like immunoreactive (LI) material was detected by western blot in homogenates of TG and medulla oblongata of subjects at prenatal and adult stages of life. Immunohistochemistry showed that expression of the TRPV1 receptor is mostly restricted to the small- and medium-sized TG neurons and to the caudal subdivision of the spinal trigeminal nucleus (Sp5C). The extent of the TRPV1-LI TG neuronal subpopulation was greater in subjects at early perinatal age than at late perinatal age and in postnatal life. Centrally, the TRPV1 receptor localized to fibre tracts and punctate elements, which were mainly distributed in the spinal tract, lamina I and inner lamina II of the Sp5C, whereas stained cells were rare. The TRPV1 receptor colocalized partially with CGRP and SP in the TG, and was incompletely codistributed with both neuropeptides in the spinal tract and in the superficial laminae of the Sp5C. Substantial differences were noted with respect to the distribution of the TRPV1-LI structures described in the rat Sp5C and with respect to the temporal expression of the receptor during the development of the rat spinal dorsal horn. The distinctive localization of TRPV1-LI material supports the concept of the involvement of TRPV1 receptor in the functional activity of the protopathic compartment of the human trigeminal sensory system, i.e. the processing and neurotransmission of thermal and pain stimuli.

The human cuneate nucleus contains discrete subregions whose neurochemical features match those of the relay nuclei for nociceptive information.

The present paper is aimed at defining distinctive subdivisions of the human cuneate nucleus (Cu), evident from prenatal to old life, whose occurrence has never been clearly formalized in the human brain, or described in other species so far. It extends our early observations on the presence of gray matter areas that host strong substance P (SP) immunoreactivity in the territory of the human Cu and adjacent cuneate fascicle. Here we provide a three-dimensional reconstruction of the Cu fields rich in SP and further identify those areas by means of their immunoreactivity to the neuropeptides SP, calcitonin gene-related peptide, methionine- and leucine-enkephalin, peptide histidine-isoleucine, somatostatin and galanin, to the trophins glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor, and to the neuroplasticity proteins polysialylated neural cell adhesion molecule and growth-associated protein-43. The presence, density and distribution of immunoreactivity for each of these molecules closely resemble those occurring in the superficial layers of the caudal spinal trigeminal nucleus (Sp5C). Myelin and Nissl stainings suggest that those Cu subregions and the Sp5C superficial layers share a similar histological aspect. This work establishes the existence of definite subregions, localized within the Cu territory, that bear the neurochemical and histological features of sensory nuclei committed to the neurotransmission of protopathic stimuli, including pain. These findings appear of particular interest when considering that functional, preclinical and clinical studies show that the dorsal column nuclei, classical relay station of fine somatic tactile and proprioceptive sensory stimuli, are also involved in pain neurotransmission.