Intrathecal (IT) delivery and pharmacology of antisense oligonucleotides (ASOs) for the CNS have been successfully developed to treat spinal muscular atrophy. However, ASO pharmacokinetic (PK) and pharmacodynamic (PD) properties remain poorly understood in the IT compartment. We applied multimodal imaging techniques to elucidate the IT PK and PD of unlabeled, radioactively labeled, or fluorescently labeled ASOs targeting ubiquitously expressed or neuron-specific RNAs. Following lumbar IT bolus injection in rats, all ASOs spread rostrally along the neuraxis, adhered to meninges, and were partially cleared to peripheral lymph nodes and kidneys. Rapid association with the pia and arterial walls preceded passage of ASOs across the glia limitans, along arterial intramural basement membranes, and along white-matter axonal bundles. Several neuronal and glial cell types accumulated ASOs over time, with evidence of probable glial accumulation preceding neuronal uptake. IT doses of anti-GluR1 and anti-Gabra1 ASOs markedly reduced the mRNA and protein levels of their respective neurotransmitter receptor protein targets by 2 weeks and anti-Gabra1 ASOs also reduced binding of the GABAA receptor PET ligand 18F-flumazenil in the brain over 4 weeks. Our multimodal imaging approaches elucidate multiple transport routes underlying the CNS distribution, clearance, and efficacy of IT-dosed ASOs.
Curt Mazur, Berit Powers, Kenneth Zasadny, Jenna M. Sullivan, Hemi Dimant, Fredrik Kamme, Jacob Hesterman, John Matson, Michael Oestergaard, Marc Seaman, Robert W. Holt, Mohammed Qutaish, Ildiko Polyak, Richard Coelho, Vijay Gottumukkala, Carolynn M. Gaut, Marc Berridge, Nazira J. Albargothy, Louise Kelly, Roxana O. Carare, Jack Hoppin, Holly Kordasiewicz, Eric E. Swayze, Ajay Verma
Efferocytosis, or phagocytic clearance of dead/dying cells by brain-resident microglia and/or infiltrating macrophages, is instrumental for inflammation resolution and restoration of brain homeostasis after stroke. Here, we identify the signal transducer and activator of transcription 6/arginase1 (STAT6/Arg1) signaling axis as a potentially novel mechanism that orchestrates microglia/macrophage responses in the ischemic brain. Activation of STAT6 was observed in microglia/macrophages in the ischemic territory in a mouse model of stroke and in stroke patients. STAT6 deficiency resulted in reduced clearance of dead/dying neurons, increased inflammatory gene signature in microglia/macrophages, and enlarged infarct volume early after experimental stroke. All of these pathological changes culminated in an increased brain tissue loss and exacerbated long-term functional deficits. Combined in vivo analyses using BM chimeras and in vitro experiments using microglia/macrophage-neuron cocultures confirmed that STAT6 activation in both microglia and macrophages was essential for neuroprotection. Adoptive transfer of WT macrophages into STAT6-KO mice reduced accumulation of dead neurons in the ischemic territory and ameliorated brain infarction. Furthermore, decreased expression of Arg1 in STAT6–/– microglia/macrophages was responsible for impairments in efferocytosis and loss of antiinflammatory modality. Our study suggests that efferocytosis via STAT6/Arg1 modulates microglia/macrophage phenotype, accelerates inflammation resolution, and improves stroke outcomes.
Wei Cai, Xuejiao Dai, Jie Chen, Jingyan Zhao, Mingyue Xu, Lili Zhang, Boyu Yang, Wenting Zhang, Marcelo Rocha, Toshimasa Nakao, Julia Kofler, Yejie Shi, R. Anne Stetler, Xiaoming Hu, Jun Chen
Alcohol withdrawal (AW) after chronic alcohol exposure produces a series of symptoms, with AW-associated seizures being among the most serious and dangerous. However, the mechanism underlying AW seizures has yet to be established. In our mouse model, a sudden AW produced 2 waves of seizures: the first wave includes a surge of multiple seizures that occurs within hours to days of AW, and the second wave consists of sustained expression of epileptiform spikes and wave discharges (SWDs) during a protracted period of abstinence. We revealed that the structural and functional adaptations in newborn dentate granule cells (DGCs) in the hippocampus underlie the second wave of seizures but not the first wave. While the general morphology of newborn DGCs remained unchanged, AW increased the dendritic spine density of newborn DGCs, suggesting that AW induced synaptic connectivity of newborn DGCs with excitatory afferent neurons and enhanced excitability of newborn DGCs. Indeed, specific activation and suppression of newborn DGCs by the chemogenetic DREADD method increased and decreased the expression of epileptiform SWDs, respectively, during abstinence. Thus, our study unveiled that the pathological plasticity of hippocampal newborn DGCs underlies AW seizures during a protracted period of abstinence, providing critical insight into hippocampal neural circuits as a foundation to understand and treat AW seizures.
Daehoon Lee, Balu Krishnan, Hai Zhang, Hee Ra Park, Eun Jeoung Ro, Yu-Na Jung, Hoonkyo Suh
Accumulation of lysosomal storage material and late-stage neurodegeneration are hallmarks of lysosomal storage disorders (LSDs) affecting the brain. Yet, for most LSDs, including CLN3 disease, the most common form of childhood dementia, it is unclear what mechanisms drive neurologic symptoms. Do deficits arise from loss of function of the mutated protein or toxicity from storage accumulation? Here, using in vitro voltage sensitive dye imaging and in vivo electrophysiology, we find progressive hippocampal dysfunction occurs prior to notable lysosomal storage and neuronal loss in two CLN3 disease mouse models. Pharmacologic reversal of lysosomal storage deposition in young mice does not rescue this circuit dysfunction. Additionally, we find that CLN3 disease mice lose an electrophysiologic marker of new memory encoding – hippocampal sharp wave ripples. This discovery, which is also seen in Alzheimer’s disease, suggests the possibility of a shared electrophysiologic signature of dementia. Overall, our data describes new insights into previously unknown network-level changes occurring in LSDs affecting the central nervous system, and highlight the need for new therapeutic interventions targeting early circuit defects.
Rebecca C. Ahrens-Nicklas, Luis Tecedor, Arron Hall, Elena Lysenko, Akiva S. Cohen, Beverly L. Davidson, Eric D. Marsh
Itch induces scratching that removes irritants from the skin, whereas pain initiates withdrawal or avoidance of tissue damage. Whilst pain arises from both the skin and viscera, we investigated whether pruritogenic irritant mechanisms also function within visceral pathways. We show that subsets of colon-innervating sensory neurons in mice express, either individually or in combination, the pruritogenic receptors Tgr5 and the Mas-gene-related G protein-coupled receptors, Mrgpra3 and Mrgpra11. Agonists of these receptors activated subsets of colonic sensory neurons and evoked colonic afferent mechanical hypersensitivity via a TRPA1-dependent mechanism. In vivo intra-colonic administration of individual TGR5, MRGPRA3, or MRGPRC11 agonists induced pronounced visceral hypersensitivity to colorectal distension. Co-administration of these agonists as an ‘itch cocktail’ augmented hypersensitivity to colorectal distension and changed mouse behaviour. These irritant mechanisms were maintained and enhanced in a model of chronic visceral hypersensitivity relevant to irritable bowel syndrome. Neurons from human dorsal root ganglia also expressed TGR5 as well as the human ortholog MRGPRX1 and showed increased responsiveness to pruritogenic agonists in pathological states. These data support the existence of an irritant-sensing system in the colon that is a visceral representation of the itch pathways found in skin, thereby contributing to sensory disturbances accompanying common intestinal disorders.
Joel Castro, Andrea M. Harrington, TinaMarie Lieu, Sonia Garcia-Caraballo, Jessica Maddern, Gudrun Schober, Tracey O'Donnell, Luke Grundy, Amanda L. Lumsden, Paul E. Miller, Andre Ghetti, Martin S. Steinhoff, Daniel P. Poole, Xinzhong Dong, Lin Chang, Nigel W. Bunnett, Stuart M. Brierley
The control of voluntary skeletal muscle contraction relies on action potentials, which send signals from the motor neuron through the neuromuscular junction (NMJ). Although dysfunction of the NMJ causes various neuromuscular diseases, a reliable in vitro system for disease modeling is currently unavailable. Here, we present a potentially novel 2-step, self-organizing approach for generating in vitro human NMJs from human induced pluripotent stem cells. Our simple and robust approach results in a complex NMJ structure that includes functional connectivity, recapitulating in vivo synapse formation. We used these in vitro NMJs to model the pathological features of spinal muscular atrophy, revealing the developmental and functional defects of NMJ formation and NMJ-dependent muscular contraction. Our differentiation system is therefore useful for investigating and understanding the physiology and pathology of human NMJs.
Chuang-Yu Lin, Michiko Yoshida, Li-Tzu Li, Akihiro Ikenaka, Shiori Oshima, Kazuhiro Nakagawa, Hidetoshi Sakurai, Eriko Matsui, Tatsutoshi Nakahata, Megumu K. Saito
Deficiency of arginase is associated with hyperargininemia, and prominent features include spastic diplegia/tetraplegia, clonus, and hyperreflexia; loss of ambulation, intellectual disability and progressive neurological decline are other signs. To gain greater insight into the unique neuromotor features, we performed gene expression profiling of the motor cortex of a murine model of the disorder. Coexpression network analysis suggested an abnormality with myelination, which was supported by limited existing human data. Utilizing electron microscopy, marked dysmyelination was detected in 2-week-old homozygous Arg1-KO mice. The corticospinal tract was found to be adversely affected, supporting dysmyelination as the cause of the unique neuromotor features and implicating oligodendrocyte impairment in a deficiency of hepatic Arg1. Following neonatal hepatic gene therapy to express Arg1, the subcortical white matter, pyramidal tract, and corticospinal tract all showed a remarkable recovery in terms of myelinated axon density and ultrastructural integrity with active wrapping of axons by nearby oligodendrocyte processes. These findings support the following conclusions: arginase deficiency is a leukodystrophy affecting the brain and spinal cord while sparing the peripheral nervous system, and neonatal AAV hepatic gene therapy can rescue the defects associated with myelinated axons, strongly implicating the functional recovery of oligodendrocytes after restoration of hepatic arginase activity.
Xiao-Bo Liu, Jillian R. Haney, Gloria Cantero, Jenna R. Lambert, Marcos Otero-Garcia, Brian Truong, Andrea Gropman, Inma Cobos, Stephen D. Cederbaum, Gerald S. Lipshutz
Chemotherapy-induced peripheral neuropathy is one of the most prevalent dose-limiting toxicities of anticancer therapy. Development of effective therapies to prevent chemotherapy-induced neuropathies could be enabled by a mechanistic understanding of axonal breakdown following exposure to neuropathy-causing agents. Here, we reveal the molecular mechanisms underlying axon degeneration induced by 2 widely used chemotherapeutic agents with distinct mechanisms of action: vincristine and bortezomib. We showed previously that genetic deletion of SARM1 blocks vincristine-induced neuropathy and demonstrate here that it also prevents axon destruction following administration of bortezomib in vitro and in vivo. Using cultured neurons, we found that vincristine and bortezomib converge on a core axon degeneration program consisting of nicotinamide mononucleotide NMNAT2, SARM1, and loss of NAD+ but engage different upstream mechanisms that closely resemble Wallerian degeneration after vincristine and apoptosis after bortezomib. We could inhibit the final common axon destruction pathway by preserving axonal NAD+ levels or expressing a candidate gene therapeutic that inhibits SARM1 in vitro. We suggest that these approaches may lead to therapies for vincristine- and bortezomib-induced neuropathies and possibly other forms of peripheral neuropathy.
Stefanie Geisler, Ryan A. Doan, Galen C. Cheng, Aysel Cetinkaya-Fisgin, Shay X. Huang, Ahmet Höke, Jeffrey Milbrandt, Aaron DiAntonio
Chromatin modifiers act to coordinate gene expression changes critical to neuronal differentiation from neural stem/progenitor cells (NSPCs). Lysine-specific methyltransferase 2D (KMT2D) encodes a histone methyltransferase that promotes transcriptional activation, and is frequently mutated in cancers and in the majority (>70%) of patients diagnosed with the congenital, multisystem intellectual disability (ID) disorder Kabuki syndrome 1 (KS1). Critical roles for KMT2D are established in various non-neural tissues, but the effects of KMT2D loss in brain cell development have not been described. We conducted parallel studies of proliferation, differentiation, transcription, and chromatin profiling in KMT2D-deficient human and mouse models to define KMT2D-regulated functions in neurodevelopmental contexts, including adult-born hippocampal NSPCs in vivo and in vitro. We report cell-autonomous defects in proliferation, cell cycle, and survival, accompanied by early NSPC maturation in several KMT2D-deficient model systems. Transcriptional suppression in KMT2D-deficient cells indicated strong perturbation of hypoxia-responsive metabolism pathways. Functional experiments confirmed abnormalities of cellular hypoxia responses in KMT2D-deficient neural cells, and accelerated NSPC maturation in vivo. Together, our findings support a model in which loss of KMT2D function suppresses expression of oxygen-responsive gene programs important to neural progenitor maintenance, resulting in precocious neuronal differentiation in a mouse model of KS1.
Giovanni A. Carosso, Leandros Boukas, Jonathan J. Augustin, Ha Nam Nguyen, Briana L. Winer, Gabrielle H. Cannon, Johanna D. Robertson, Li Zhang, Kasper D. Hansen, Loyal A. Goff, Hans T. Bjornsson
There is increased interest in whether bariatric surgeries such as Roux-en-Y gastric bypass (RYGB) achieve their profound weight-lowering effects in morbidly obese individuals through the brain. Hypothalamic inflammation is a well-recognized etiologic factor in obesity pathogenesis and so represents a potential target of RYGB, but clinical evidence in support of this is limited. We therefore assessed hypothalamic T2-weighted signal intensities (T2W SI) and fractional anisotropy (FA) values, two validated radiologic measures of brain inflammation, in relation to BMI and fat mass as well as circulating inflammatory (C-reactive peptide - CrP) and metabolic markers in a cohort of 27 RYGB patients at baseline, 6 months and 12 months after surgery. We found that RYGB progressively increased hypothalamic T2W SI values while it progressively decreased hypothalamic FA values. Regression analyses further revealed that this could be most strongly linked to plasma CrP levels which independently predicted hypothalamic FA values when adjusting for age, sex, fat mass and diabetes diagnosis. These findings suggest that RYGB has a major time-dependent impact on hypothalamic inflammation status possibly by attenuating peripheral inflammation. They also suggest that hypothalamic FA values may provide a more specific radiologic measure of hypothalamic inflammation than more commonly used T2W SI values.
Mohammed K. Hankir, Michael Rullmann, Florian Seyfried, Sven Preusser, Sindy Poppitz, Stefanie Heba, Kostantinos Gousias, Jana Hoyer, Tatjana Schütz, Arne Dietrich, Karsten Müller, Burkhard Pleger
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