DEP domain–containing 5 protein (DEPDC5) is a repressor of the recently recognized amino acid–sensing branch of the mTORC1 pathway. So far, its function in the brain remains largely unknown. Germline loss-of-function mutations in DEPDC5 have emerged as a major cause of familial refractory focal epilepsies, with case reports of sudden unexpected death in epilepsy (SUDEP). Remarkably, a fraction of patients also develop focal cortical dysplasia (FCD), a neurodevelopmental cortical malformation. We therefore hypothesized that a somatic second-hit mutation arising during brain development may support the focal nature of the dysplasia. Here, using postoperative human tissue, we provide the proof of concept that a biallelic 2-hit — brain somatic and germline — mutational mechanism in DEPDC5 causes focal epilepsy with FCD. We discovered a mutation gradient with a higher rate of mosaicism in the seizure-onset zone than in the surrounding epileptogenic zone. Furthermore, we demonstrate the causality of a Depdc5 brain mosaic inactivation using CRISPR-Cas9 editing and in utero electroporation in a mouse model recapitulating focal epilepsy with FCD and SUDEP-like events. We further unveil a key role of Depdc5 in shaping dendrite and spine morphology of excitatory neurons. This study reveals promising therapeutic avenues for treating drug-resistant focal epilepsies with mTORC1-targeting molecules.
Théo Ribierre, Charlotte Deleuze, Alexandre Bacq, Sara Baldassari, Elise Marsan, Mathilde Chipaux, Giuseppe Muraca, Delphine Roussel, Vincent Navarro, Eric Leguern, Richard Miles, Stéphanie Baulac
Spinal muscular atrophy (SMA), a degenerative motor neuron (MN) disease caused by loss of functional SMN protein due to SMN1 gene mutations, is a leading cause of infant mortality. Increasing SMN levels ameliorates the disease phenotype and is unanimously accepted as a therapeutic approach for SMA patients. The ubiquitin/proteasome system is known to regulate SMN protein levels; however whether autophagy controls SMN levels remains poorly explored. Here we show that SMN protein is degraded by autophagy. Pharmacological and genetic inhibition of autophagy increase SMN levels, while induction of autophagy decreases SMN. SMN degradation occurs via its interaction with the autophagy adapter p62/SQSTM1. We also show that SMA neurons display reduced autophagosome clearance, increased p62/ubiquitinated protein levels, and hyperactivated mTORC1 signaling. Importantly, reducing p62 levels markedly increases SMN and its binding partner gemin2, promotes MN survival and extends lifespan in fly and mouse SMA models revealing p62 as a new potential therapeutic target to treat SMA.
Natalia Rodriguez-Muela, Andrey Parkhitko, Tobias Grass, Rebecca M. Gibbs, Erika M. Norabuena, Norbert Perrimon, Rajat Singh, Lee L. Rubin
Complications of diabetes affect tissues throughout body, including central nervous system. Epidemiological studies show that diabetic patients have increased risk of depression, anxiety, age-related cognitive decline and Alzheimer’s disease. Mice lacking insulin receptor in brain or on hypothalamic neurons display an array of metabolic abnormalities, however, the role of insulin action on astrocytes and neurobehaviors remains less well-studied. Here, we demonstrate that astrocytes are a direct insulin target in the brain and that knockout of IR on astrocytes causes increased anxiety and depressive-like behaviors in mice. This can be reproduced in part by deletion of IR on astrocytes in the nucleus accumbens. At a molecular level, loss of insulin signaling in astrocytes impaired tyrosine phosphorylation of Munc18c. This led to decreased exocytosis of ATP from astrocytes, resulting in decreased purinergic signaling on dopaminergic neurons. These reductions contributed to decreased dopamine release from brain slices. Central administration of ATP analogues could reverse depressive-like behaviors in mice with astrocyte IR knockout. Thus, astrocytic insulin signaling plays an important role in dopaminergic signaling, providing a potential mechanism by which astrocytic insulin action may contribute to increased rates of depression in people with diabetes, obesity and other insulin resistant states.
Weikang Cai, Chang Xue, Masaji Sakaguchi, Masahiro Konishi, Alireza Shirazian, Heather A. Ferris, Mengyao Li, Ruichao Yu, Andre Kleinridders, Emmanuel N. Pothos, C. Ronald Kahn
Cerebral white matter injury (WMI) persistently disrupts myelin regeneration by oligodendrocyte progenitor cells (OPCs). We identified a specific bioactive hyaluronan fragment (bHAf) that downregulates myelin gene expression and chronically blocks OPC maturation and myelination via a tolerance-like mechanism that dysregulates pro-myelination signaling via AKT. Desensitization of AKT occurs via TLR4 but not TLR2 or CD44. OPC differentiation was selectively blocked by bHAf in a maturation-dependent fashion at the late OPC (preOL) stage by a noncanonical TLR4/TRIF pathway that induced persistent activation of the FoxO3 transcription factor downstream of AKT. Activated FoxO3 selectively localized to oligodendrocyte lineage cells in white matter lesions from human preterm neonates and adults with multiple sclerosis. FoxO3 constraint of OPC maturation was bHAf dependent, and involved interactions at the FoxO3 and MBP promoters with the chromatin remodeling factor Brg1 and the transcription factor Olig2, which regulate OPC differentiation. WMI has adapted an immune tolerance–like mechanism whereby persistent engagement of TLR4 by bHAf promotes an OPC niche at the expense of myelination by engaging a FoxO3 signaling pathway that chronically constrains OPC differentiation.
Taasin Srivastava, Parham Diba, Justin M. Dean, Fatima Banine, Daniel Shaver, Matthew Hagen, Xi Gong, Weiping Su, Ben Emery, Daniel L. Marks, Edward N. Harris, Bruce Baggenstoss, Paul H. Weigel, Larry S. Sherman, Stephen A. Back
Activation of non-neuronal microglia is thought to play a causal role in spinal processing of neuropathic pain. To specifically investigate microglia-mediated effects in a model of neuropathic pain and overcome methodological limitations of previous approaches exploring microglia function upon nerve injury, we selectively ablated resident microglia by intracerebroventricular (icv) ganciclovir infusion into male CD11b-HSVTK transgenic mice, which was followed by a rapid, complete and persistent (23 weeks) repopulation of the CNS by peripheral myeloid cells. In repopulated mice that underwent sciatic nerve injury, we observed a normal response to mechanical stimuli, but an absence of thermal hypersensitivity ipsilateral to the injured nerve. Furthermore, we found that neuronal expression of calcitonin gene-related peptide (CGRP), which is a marker of neurons essential for heat responses, was diminished in the dorsal horn of the spinal cord in repopulated mice. These findings demonstrate distinct mechanisms for heat and mechanical hypersensitivity, highlighting a crucial contribution of CNS myeloid cells in the facilitation of noxious heat.
Stefanie Kälin, Kelly R. Miller, Roland E. Kälin, Marina Jendrach, Christian Witzel, Frank L. Heppner
Rasmussen’s encephalitis (RE) is a chronic inflammatory brain disorder that causes frequent seizures and unilateral hemispheric atrophy with progressive neurological deficits. Hemispherectomy remains the only treatment that leads to seizure freedom for this refractory epileptic syndrome. The absence of an animal model of disease has been a major obstacle hampering the development of effective therapies. Here, we describe an experimental mouse model that shares several clinical and pathological features with the human disease. Immunodeficient mice injected with peripheral blood mononuclear cells from RE patients and monitored by video electroencephalography developed severe seizures of cortical origin and showed intense astrogliosis and accumulation of human IFN-γ– and granzyme B–expressing T lymphocytes in the brain compared with mice injected with immune cells from control subjects. We also provide evidence for the efficacy of α4 integrin blockade, an approved therapy for the treatment of multiple sclerosis and Crohn’s disease, in reducing inflammatory markers associated with RE in the CNS. This model holds promise as a valuable tool for understanding the pathology of RE and for developing patient-tailored experimental therapeutics.
Hania Kebir, Lionel Carmant, François Fontaine, Kathie Béland, Ciprian M. Bosoi, Nathalie T. Sanon, Jorge I. Alvarez, Sébastien Desgent, Camille L. Pittet, David Hébert, Marie-Josée Langlois, Rose-Marie Rébillard, Dang K. Nguyen, Cécile Cieuta-Walti, Gregory L. Holmes, Howard P. Goodkin, John R. Mytinger, Mary B. Connolly, Alexandre Prat, Elie Haddad
The apolipoprotein E E4 allele of the APOE gene is the strongest genetic factor for late-onset Alzheimer disease (LOAD). There is compelling evidence that apoE influences Alzheimer disease (AD) in large part by affecting amyloid β (Aβ) aggregation and clearance; however, the molecular mechanism underlying these findings remains largely unknown. Herein, we tested whether anti–human apoE antibodies can decrease Aβ pathology in mice producing both human Aβ and apoE4, and investigated the mechanism underlying these effects. We utilized APPPS1-21 mice crossed to apoE4-knockin mice expressing human apoE4 (APPPS1-21/APOE4). We discovered an anti–human apoE antibody, anti–human apoE 4 (HAE-4), that specifically recognizes human apoE4 and apoE3 and preferentially binds nonlipidated, aggregated apoE over the lipidated apoE found in circulation. HAE-4 also binds to apoE in amyloid plaques in unfixed brain sections and in living APPPS1-21/APOE4 mice. When delivered centrally or by peripheral injection, HAE-4 reduced Aβ deposition in APPPS1-21/APOE4 mice. Using adeno-associated virus to express 2 different full-length anti–apoE antibodies in the brain, we found that HAE antibodies decreased amyloid accumulation, which was dependent on Fcγ receptor function. These data support the hypothesis that a primary mechanism for apoE-mediated plaque formation may be a result of apoE aggregation, as preferentially targeting apoE aggregates with therapeutic antibodies reduces Aβ pathology and may represent a selective approach to treat AD.
Fan Liao, Aimin Li, Monica Xiong, Nga Bien-Ly, Hong Jiang, Yin Zhang, Mary Beth Finn, Rosa Hoyle, Jennifer Keyser, Katheryn B. Lefton, Grace O. Robinson, Javier Remolina Serrano, Adam P. Silverman, Jing L. Guo, Jennifer Getz, Kirk Henne, Cheryl E.G. Leyns, Gilbert Gallardo, Jason D. Ulrich, Patrick M. Sullivan, Eli Paul Lerner, Eloise Hudry, Zachary K. Sweeney, Mark S. Dennis, Bradley T. Hyman, Ryan J. Watts, David M. Holtzman
Loss of bladder control is a challenging outcome facing patients with spinal cord injury (SCI). We report that systemic blocking of pro–nerve growth factor (proNGF) signaling through p75 with a CNS-penetrating small-molecule p75 inhibitor resulted in significant improvement in bladder function after SCI in rodents. The usual hyperreflexia was attenuated with normal bladder pressure, and automatic micturition was acquired weeks earlier than in the controls. The improvement was associated with increased excitatory input to the spinal cord, in particular onto the tyrosine hydroxylase–positive fibers in the dorsal commissure. The drug also had an effect on the bladder itself, as the urothelial hyperplasia and detrusor hypertrophy that accompany SCI were largely prevented. Urothelial cell loss that precedes hyperplasia was dependent on p75 in response to urinary proNGF that is detected after SCI in rodents and humans. Surprisingly, death of urothelial cells and the ensuing hyperplastic response were beneficial to functional recovery. Deleting p75 from the urothelium prevented urothelial death, but resulted in reduction in overall voiding efficiency after SCI. These results unveil a dual role of proNGF/p75 signaling in bladder function under pathological conditions with a CNS effect overriding the peripheral one.
Jae Cheon Ryu, Katharine Tooke, Susan E. Malley, Anastasia Soulas, Tirzah Weiss, Nisha Ganesh, Nabila Saidi, Stephanie Daugherty, Uri Saragovi, Youko Ikeda, Irina Zabbarova, Anthony J. Kanai, Mitsuharu Yoshiyama, H. Francis Farhadi, William C. de Groat, Margaret A. Vizzard, Sung Ok Yoon
During development, Sox2 is indispensable for cell division and differentiation, yet its roles in regenerating tissues are less clear. Here, we used combinations of transgenic mouse models to reveal that Sox2 haploinsufficiency (Sox2haplo) increases rather than impairs cochlear regeneration in vivo. Sox2haplo cochleae had delayed terminal mitosis and ectopic sensory cells, yet normal auditory function. Sox2haplo amplified and expanded domains of damage-induced Atoh1+ transitional cell formation in neonatal cochlea. Wnt activation via β-catenin stabilization (β-cateninGOF) alone failed to induce proliferation or transitional cell formation. By contrast, β-cateninGOF caused proliferation when either Sox2haplo or damage was present, and transitional cell formation when both were present in neonatal, but not mature, cochlea. Mechanistically, Sox2haplo or damaged neonatal cochleae showed lower levels of Sox2 and Hes5, but not of Wnt target genes. Together, our study unveils an interplay between Sox2 and damage in directing tissue regeneration and Wnt responsiveness and thus provides a foundation for potential combinatorial therapies aimed at stimulating mammalian cochlear regeneration to reverse hearing loss in humans.
Patrick J. Atkinson, Yaodong Dong, Shuping Gu, Wenwen Liu, Elvis Huarcaya Najarro, Tomokatsu Udagawa, Alan G. Cheng
Painful diabetic neuropathy (PDN) is an intractable complication of diabetes that affects 25% of patients. PDN is characterized by neuropathic pain and small-fiber degeneration, accompanied by dorsal root ganglion (DRG) nociceptor hyperexcitability and loss of their axons within the skin. The molecular mechanisms underlying DRG nociceptor hyperexcitability and small-fiber degeneration in PDN are unknown. We hypothesize that chemokine CXCL12/CXCR4 signaling is central to this mechanism, as we have shown that CXCL12/CXCR4 signaling is necessary for the development of mechanical allodynia, a pain hypersensitivity behavior common in PDN. Focusing on DRG neurons expressing the sodium channel Nav1.8, we applied transgenic, electrophysiological, imaging, and chemogenetic techniques to test this hypothesis. In the high-fat diet mouse model of PDN, we were able to prevent and reverse mechanical allodynia and small-fiber degeneration by limiting CXCR4 signaling or neuronal excitability. This study reveals that excitatory CXCR4/CXCL12 signaling in Nav1.8-positive DRG neurons plays a critical role in the pathogenesis of mechanical allodynia and small-fiber degeneration in a mouse model of PDN. Hence, we propose that targeting CXCR4-mediated DRG nociceptor hyperexcitability is a promising therapeutic approach for disease-modifying treatments for this currently intractable and widespread affliction.
Nirupa D. Jayaraj, Bula J. Bhattacharyya, Abdelhak A. Belmadani, Dongjun Ren, Craig A. Rathwell, Sandra Hackelberg, Brittany E. Hopkins, Herschel R. Gupta, Richard J. Miller, Daniela M. Menichella