Gene therapy approaches are being deployed to treat recessive genetic disorders by restoring the expression of mutated genes. However, the feasibility of these approaches for dominantly-inherited diseases—where treatment may require reduction in the expression of a toxic mutant protein resulting from a gain-of-function (GoF) allele—is unclear. Here we show the efficacy of allele-specific RNAi as a potential therapeutic for Charcot-Marie-Tooth type 2D (CMT2D), caused by dominant mutations in glycyl tRNA-synthetase (GARS). A de novo mutation in GARS was identified in a patient with a severe peripheral neuropathy, and a mouse model precisely recreating the mutation was produced. These mice developed a neuropathy by 3-4 weeks-of-age, validating the pathogenicity of the mutation. RNAi sequences targeting mutant GARS mRNA, but not wild-type, were optimized and then packaged into AAV9 for in vivo delivery. This almost completely prevented the neuropathy in mice treated at birth. Delaying treatment until after disease onset showed modest benefit, though this effect decreased the longer treatment was delayed. These outcomes were reproduced in a second mouse model of CMT2D using a vector specifically targeting that allele. The effects were dose dependent, and persisted for at least one year. Our findings demonstrate the feasibility of AAV9-mediated allele-specific knockdown and provide proof-of-concept for gene therapy approaches for dominant neuromuscular diseases.
Kathryn H. Morelli, Laurie B. Griffin, Nettie K. Pyne, Lindsay M. Wallace, Allison M. Fowler, Stephanie N. Oprescu, Ryuichi Takase, Na Wei, Rebecca Meyer-Schuman, Dattatreya Mellacheruvu, Jacob O. Kitzman, Samuel G. Kocen, Timothy J. Hines, Emily L. Spaulding, James R. Lupski, Alexey Nesvizhskii, Pedro Mancias, Ian J. Butler, Xiang-Lei Yang, Ya-Ming Hou, Anthony Antonellis, Scott Q. Harper, Robert W. Burgess
Arcuate nucleus agouti-related peptide (AgRP) neurons play a central role in feeding and are under complex regulation by both homeostatic hormonal and nutrient signals and hypothalamic neuronal pathways. Feeding may also be influenced by environmental cues, sensory inputs and other behaviors implying the involvement of higher brain regions. However, whether such pathways modulate feeding through direct synaptic control of AgRP neuron activity is unknown. Here we show that nociceptin-expressing neurons in the anterior bed nuclei of the stria terminalis (aBNST) make direct GABAergic inputs onto AgRP neurons. We found that activation of these neurons inhibited AgRP neurons and feeding. Activity of these neurons increased upon food availability and their ablation resulted in obesity. Furthermore, these neurons received afferent inputs from a range of upstream brain regions as well as hypothalamic nuclei. Therefore, aBNST nociceptin/GABAergic neurons may act as a gateway to feeding behavior by connecting AgRP neurons to both homeostatic and non-homeostatic neuronal inputs.
Mark A. Smith, Agharul I. Choudhury, Justyna A. Glegola, Paulius Viskaitis, Elaine E. Irvine, Pedro Caldas Custodio de Campos Silva, Sanjay Khadayate, Hanns Ulrich Zeilhofer, Dominic J. Withers
Parkinson’s disease (PD) is a common neurodegenerative disease that lacks therapies to prevent progressive neurodegeneration. Impaired energy metabolism and reduced ATP levels are common features of PD. Previous studies revealed that terazosin (TZ) enhances the activity of phosphoglycerate kinase 1 (PGK1), thereby stimulating glycolysis and increasing cellular ATP levels. Therefore, we asked whether enhancement of PGK1 activity would change the course of PD. In toxin-induced and genetic PD models in mice, rats, flies, and induced pluripotent stem cells, TZ increased brain ATP levels and slowed or prevented neuron loss. The drug increased dopamine levels and partially restored motor function. Because TZ is prescribed clinically, we also interrogated 2 distinct human databases. We found slower disease progression, decreased PD-related complications, and a reduced frequency of PD diagnoses in individuals taking TZ and related drugs. These findings suggest that enhancing PGK1 activity and increasing glycolysis may slow neurodegeneration in PD.
Rong Cai, Yu Zhang, Jacob E. Simmering, Jordan L. Schultz, Yuhong Li, Irene Fernandez-Carasa, Antonella Consiglio, Angel Raya, Philip M. Polgreen, Nandakumar S. Narayanan, Yanpeng Yuan, Zhiguo Chen, Wenting Su, Yanping Han, Chunyue Zhao, Lifang Gao, Xunming Ji, Michael J. Welsh, Lei Liu
Angelman syndrome (AS) is a neurodevelopmental disorder characterized by intellectual disability, lack of speech, ataxia, EEG abnormalities, and epilepsy. Seizures in AS individuals are common, debilitating, and often drug-resistant. Therefore, there is an unmet need for better treatment options. Cannabidiol (CBD), a major phytocannabinoid constituent of cannabis, has antiseizure activity and behavioral benefits in preclinical and clinical studies for some disorders associated with epilepsy, suggesting that the same could be true for AS. Here we show that acute CBD (100 mg/kg) attenuated hyperthermia- and acoustically-induced seizures in a mouse model of AS. However, neither acute CBD nor a two-weeklong course of CBD administered immediately after a kindling protocol could halt the pro-epileptogenic plasticity observed in AS model mice. CBD had a dose-dependent sedative effect, but did not have an impact on motor performance. CBD abrogated the enhanced intracortical local field potential power, including delta and theta rhythms observed in AS model mice, indicating that CBD administration could also help normalize the EEG deficits observed in individuals with AS. Our results provide critical preclinical evidence supporting CBD treatment of seizures and alleviation of EEG abnormalities in AS, and will thus help guide the rational development of CBD as an AS treatment.
Bin Gu, Manhua Zhu, Madison R. Glass, Marie Rougié, Viktoriya D. Nikolova, Sheryl S. Moy, Paul R. Carney, Benjamin D. Philpot
Growing evidence shows that alterations occurring at early developmental stages contribute to symptoms manifested in adulthood in the setting of neurodegenerative diseases. Here, we studied the molecular mechanisms causing giant axonal neuropathy (GAN), a severe neurodegenerative disease due to loss-of-function of the gigaxonin-E3 ligase. We showed that gigaxonin governs Sonic Hedgehog (Shh) induction, the developmental pathway patterning the dorso-ventral axis of the neural tube and muscles, by controlling the degradation of the Shh-bound Patched receptor. Similarly to Shh inhibition, repression of gigaxonin in zebrafish impaired motor neuron specification and somitogenesis and abolished neuromuscular junction formation and locomotion. Shh signaling was impaired in gigaxonin null zebrafish and was corrected by both pharmacological activation of the Shh pathway and human gigaxonin, pointing to an evolutionary-conserved mechanism regulating Shh signaling. Gigaxonin-dependent inhibition of Shh activation was also demonstrated in primary fibroblasts from GAN patients and in a Shh activity reporter line depleted in gigaxonin. Our findings establish gigaxonin as a key E3 ligase that positively controls the initiation of Shh transduction, reveal the causal role of Shh dysfunction in motor deficits, thus highlighting the developmental origin of GAN.
Yoan Arribat, Karolina S Mysiak, Léa Lescouzères, Alexia Boizot, Maxime Ruiz, Mireille Rossel, Pascale Bomont
Inflammatory destruction of iron-rich myelin is characteristic of multiple sclerosis (MS). Although iron is needed for oligodendrocytes to produce myelin during development, its deposition has also been linked to neurodegeneration and inflammation, including in MS. We report perivascular iron deposition in multiple sclerosis lesions that was mirrored in 72 lesions from 13 marmosets with experimental autoimmune encephalomyelitis. Iron accumulated mainly inside microglia/macrophages from 6 weeks after demyelination. Consistently, expression of transferrin receptor, the brain’s main iron-influx protein, increased as lesions aged. Iron was uncorrelated with inflammation and postdated initial demyelination, suggesting that iron is not directly pathogenic. Iron homeostasis was at least partially restored in remyelinated, but not persistently demyelinated, lesions. Taken together, our results suggest that iron accumulation in the weeks after inflammatory demyelination may contribute to lesion repair rather than inflammatory demyelination per se.
Nathanael J. Lee, Seung-Kwon Ha, Pascal Sati, Martina Absinta, Govind Nair, Nicholas J. Luciano, Emily C. Leibovitch, Cecil C. Yen, Tracey A. Rouault, Afonso C. Silva, Steven Jacobson, Daniel S. Reich
Reactive astrocytes are associated with every form of neurological injury. Despite their ubiquity, the molecular mechanisms controlling their production and diverse functions remain poorly defined. Because many features of astrocyte development are recapitulated in reactive astrocytes, we investigated the role of nuclear factor I-A (NFIA), a key transcriptional regulator of astrocyte development whose contributions to reactive astrocytes remain undefined. Here, we show that NFIA is highly expressed in reactive astrocytes in human neurological injury and identify unique roles across distinct injury states and regions of the CNS. In the spinal cord, after white matter injury (WMI), NFIA-deficient astrocytes exhibit defects in blood-brain barrier remodeling, which are correlated with the suppression of timely remyelination. In the cortex, after ischemic stroke, NFIA is required for the production of reactive astrocytes from the subventricular zone (SVZ). Mechanistically, NFIA directly regulates the expression of thrombospondin 4 (Thbs4) in the SVZ, revealing a key transcriptional node regulating reactive astrogenesis. Together, these studies uncover critical roles for NFIA in reactive astrocytes and illustrate how region- and injury-specific factors dictate the spectrum of reactive astrocyte responses.
Dylan Laug, Teng-Wei Huang, Navish A. Bosquez Huerta, Anna Yu-Szu Huang, Debosmita Sardar, Joshua Ortiz-Guzman, Jeffrey C. Carlson, Benjamin R. Arenkiel, Chay T. Kuo, Carrie A. Mohila, Stacey M. Glasgow, Hyun Kyoung Lee, Benjamin Deneen
Excessive alcohol consumption is associated with spontaneous burning pain, hyperalgesia and allodynia. Although acetaldehyde has been implicated in the painful alcoholic neuropathy, the mechanism by which the ethanol metabolite causes pain symptoms is unknown. Acute ethanol ingestion caused delayed mechanical allodynia in mice. Inhibition of alcohol dehydrogenase (ADH) or deletion of transient receptor potential ankyrin 1 (TRPA1), a sensor for oxidative and carbonyl stress, prevented allodynia. Acetaldehyde generated by ADH in both liver and Schwann cells surrounding nociceptors was required for TRPA1-induced mechanical allodynia. Plp1-Cre;Trpa1fl/fl mice with a tamoxifen-inducible specific deletion of TRPA1 in Schwann cells revealed that channel activation by acetaldehyde in these cells initiates a NADPH oxidase-1 (NOX-1)-dependent production of hydrogen peroxide (H2O2) and 4-hydroxynonenal (4-HNE), which sustains allodynia by paracrine targeting of nociceptor TRPA1. Chronic ethanol ingestion caused prolonged mechanical allodynia and loss of intraepidermal small nerve fibers in WT mice. While Trpa1-/- or Plp1-Cre;Trpa1fl/fl mice did not develop mechanical allodynia, they did not show any protection from the small fiber neuropathy. Human Schwann cells express ADH/TRPA1/NOX1 and recapitulate the proalgesic functions of mouse Schwann cells. TRPA1 antagonists might attenuate some symptoms of alcohol-related pain.
Francesco De Logu, Simone Li Puma, Lorenzo Landini, Francesca Portelli, Alessandro Innocenti, Daniel Souza Monteiro de Araújo, Malvin N. Janal, Riccardo Patacchini, Nigel W. Bunnett, Pierangelo Geppetti, Romina Nassini
Brain somatic mutations confer genomic diversity in the human brain and cause neurodevelopmental disorders. Recently, brain somatic activating mutations in MTOR have been identified as a major etiology of intractable epilepsy in patients with cortical malformations. However, the molecular genetic mechanism of how brain somatic mutations in MTOR cause intractable epilepsy has remained elusive. In this study, translational profiling of intractable epilepsy mouse models with brain somatic mutations and genome-edited cells revealed a novel translational dysregulation mechanism and mTOR activation–sensitive targets mediated by human MTOR mutations that lead to intractable epilepsy with cortical malformation. These mTOR targets were found to be regulated by novel mTOR-responsive 5′-UTR motifs, distinct from known mTOR inhibition–sensitive targets regulated by 5′ terminal oligopyrimidine motifs. Novel mTOR target genes were validated in patient brain tissues, and the mTOR downstream effector eIF4E was identified as a new therapeutic target in intractable epilepsy via pharmacological or genetic inhibition. We show that metformin, an FDA-approved eIF4E inhibitor, suppresses intractable epilepsy. Altogether, the present study describes translational dysregulation resulting from brain somatic mutations in MTOR, as well as the pathogenesis and potential therapeutic targets of intractable epilepsy.
Jang Keun Kim, Jun Cho, Se Hoon Kim, Hoon-Chul Kang, Dong-Seok Kim, V. Narry Kim, Jeong Ho Lee
Delayed ischemic neurological deficit (DIND) is a major driver of adverse outcomes in patients with aneurysmal subarachnoid hemorrhage (aSAH) defining an unmet need for therapeutic development. Cell-free hemoglobin that is released from erythrocytes into the cerebrospinal fluid (CSF) is suggested to cause vasoconstriction and neuronal toxicity and correlates with the occurrence of DIND. Cell-free hemoglobin in the CSF of patients with aSAH disrupted dilatory NO signaling ex vivo in cerebral arteries, which shifted vascular tone balance from dilation to constriction. We found that selective removal of hemoglobin from patient CSF with a haptoglobin-affinity column or its sequestration in a soluble hemoglobin-haptoglobin complex was sufficient to restore physiological vascular responses. In a sheep model, administration of haptoglobin into the CSF inhibited hemoglobin-induced cerebral vasospasm and preserved vascular NO-signaling. We identified two pathways of hemoglobin delocalization from CSF into the brain parenchyma and into the NO-sensitive compartment of small cerebral arteries. Both pathways were critical for hemoglobin-toxicity and were interrupted by the large hemoglobin-haptoglobin complex that inhibited spatial requirements for hemoglobin reactions with NO in tissues. Collectively, our data show that compartmentalization of hemoglobin by haptoglobin provides a novel framework for innovation aimed at reducing hemoglobin-driven neurological damage after subarachnoid bleeding.
Michael Hugelshofer, Raphael M. Buzzi, Christian A. Schaer, Henning Richter, Kevin Akeret, Vania Anagnostakou, Leila Mahmoudi, Raphael Vaccani, Florence Vallelian, Jeremy W. Deuel, Peter W. Kronen, Zsolt Kulcsar, Luca Regli, Jin Hyen Baek, Ivan S. Pires, Andre F. Palmer, Matthias Dennler, Rok Humar, Paul W. Buehler, Patrick R. Kircher, Emanuela Keller, Dominik J. Schaer