Chronic pain, whether of inflammatory or neuropathic origin, affects about 18% of the population of developed countries, and most current treatments are only moderately effective and/or cause serious side effects. Therefore, the development of novel therapeutic approaches still represents a major challenge. The Na,K-ATPase modulator FXYD2 is critically required for the maintenance of neuropathic pain in rodents. Here, we set up a therapeutic protocol based on the use of chemically modified antisense oligonucleotides (ASOs) to inhibit FXYD2 expression and treat chronic pain. We identified an ASO targeting a 20-nucleotide stretch in the FXYD2 mRNA that is evolutionarily conserved between rats and humans and is a potent inhibitor of FXYD2 expression. We used this sequence to synthesize lipid-modified forms of ASO (FXYD2-LASO) to facilitate their entry into dorsal root ganglia neurons. We established that intrathecal or intravenous injections of FXYD2-LASO in rat models of neuropathic or inflammatory pain led to a virtually complete alleviation of their pain symptoms, without causing obvious side effects. Remarkably, by using 2′-O-2-methoxyethyl chemical stabilization of the ASO (FXYD2-LASO-Gapmer), we could significantly prolong the therapeutic action of a single treatment up to 10 days. This study establishes FXYD2-LASO-Gapmer administration as a promising and efficient therapeutic strategy for long-lasting relief of chronic pain conditions in human patients.
Alexandre Derre, Noelian Soler, Valentine Billoux, Sebastien Benizri, Brune Vialet, Cyril Rivat, Philippe Barthélémy, Patrick Carroll, Alexandre Pattyn, Stephanie Venteo
Spinal motor neurons have been implicated in the loss of motor function that occurs with advancing age. However, the cellular and molecular mechanisms that impair the function of these neurons during aging remain unknown. Here, we show that motor neurons do not die in old female and male mice, rhesus monkeys, and humans. Instead, these neurons selectively and progressively shed excitatory synaptic inputs throughout the soma and dendritic arbor during aging. Thus, aged motor neurons contain a motor circuitry with a reduced ratio of excitatory to inhibitory synapses that may be responsible for the diminished ability to activate motor neurons to commence movements. An examination of the motor neuron translatome (ribosomal transcripts) in male and female mice reveals genes and molecular pathways with roles in glia-mediated synaptic pruning, inflammation, axonal regeneration, and oxidative stress that are upregulated in aged motor neurons. Some of these genes and pathways are also found altered in motor neurons affected with amyotrophic lateral sclerosis (ALS) and responding to axotomy, demonstrating that aged motor neurons are under significant stress. Our findings show mechanisms altered in aged motor neurons that could serve as therapeutic targets to preserve motor function during aging.
Ryan W. Castro, Mikayla C. Lopes, Robert E. Settlage, Gregorio Valdez
Elevated blood glucose levels, or hyperglycemia, can increase brain excitability and amyloid-beta (Aβ) release offering a mechanistic link between type-2-diabetes and Alzheimer’s disease (AD). Since the cellular mechanisms governing this relationship are poorly understood, we explored whether ATP-sensitive potassium (KATP) channels, which couple changes in energy availability with cellular excitability, play a role in AD pathogenesis. First, we demonstrate that KATP channel subunits, Kir6.2/KCNJ11 and SUR1/ABCC8 are expressed on excitatory and inhibitory neurons in the human brain and cortical expression of KCNJ11 and ABCC8 changes with Alzheimer’s pathology in humans and mice. Next, we explored whether eliminating neuronal KATP channel activity uncoupled the relationship between metabolism, excitability, and Aβ pathology in a novel mouse model of cerebral amyloidosis and neuronal KATP channel ablation (e.g. APP/PS1, Kir6.2-/- mouse). Using both acute and chronic paradigms, we demonstrate that Kir6.2-KATP channels are metabolic sensors that regulate hyperglycemic-dependent increases in interstitial fluid levels of Aβ, amyloidogenic processing of APP, and amyloid plaque formation, which may be dependent on lactate release. These studies identify a new role for Kir6.2-KATP channels in Alzheimer’s disease and suggest that pharmacological manipulation of Kir6.2-KATP channels holds therapeutic promise in reducing Aβ pathology in diabetic or prediabetic patients.
John Grizzanti, William R. Moritz, Morgan C. Pait, Molly Stanley, Sarah D. Kaye, Caitlin M. Carroll, Nicholas J. Constantino, Lily J. Deitelzweig, James A. Snipes, Derek Kellar, Emily E. Caesar, Ryan J. Pettit-Mee, Stephen M. Day, Jonathon P. Sens, Noelle I. Nicol, Jasmeen Dhillon, Maria S. Remedi, Drew D. Kiraly, Celeste M. Karch, Colin G. Nichols, David M. Holtzman, Shannon L. Macauley
Myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD) is an inflammatory demyelinating central nervous system condition characterized by the presence of MOG autoantibodies. We sought to investigate whether human MOG autoantibodies are capable of mediating damage to MOG-expressing cells through multiple mechanisms. We developed high-throughput assays to measure complement activity (CA), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), and antibody-dependent cellular cytotoxicity (ADCC) of live MOG-expressing cells. MOGAD patient sera effectively mediate all of these effector functions. Our collective analyses reveal that [i] cytotoxicity is not incumbent on MOG autoantibody quantity alone, [ii] engagement of effector functions by MOGAD patient serum is bimodal, with some sera exhibiting cytotoxic capacity while others did not, [iii] the magnitude of CDC and ADCP is elevated closer to relapse, while MOG-IgG binding is not, and [iv] all IgG subclasses can damage MOG-expressing cells. Histopathology from a representative MOGAD case revealed congruence between lesion histology and serum CDC and ADCP, and we identified NK cells, mediators of ADCC, in the cerebrospinal fluid of relapsing MOGAD patients. Thus, MOGAD-derived autoantibodies are cytotoxic to MOG-expressing cells through multiple mechanisms and assays quantifying CDC and ADCP may prove to be effective tools for predicting risk of future relapses.
Soumya S. Yandamuri, Beata Filipek, Abeer H. Obaid, Nikhil Lele, Joshua M. Thurman, Naila Makhani, Richard J. Nowak, Yong Guo, Claudia F. Lucchinetti, Eoin P. Flanagan, Erin E. Longbrake, Kevin C. O'Connor
Makorin ring finger protein 3 (MKRN3) was identified as an inhibitor of puberty initiation with the report of loss-of-function mutations in association with central precocious puberty. Consistent with this inhibitory role, a prepubertal decrease in Mkrn3 expression was observed in the mouse hypothalamus. Here, we investigated the mechanisms of action of MKRN3 in the central regulation of puberty onset. We showed that MKRN3 deletion in hypothalamic neurons derived from human induced pluripotent stem cells was associated with significant changes in expression of genes controlling hypothalamic development and plasticity. Mkrn3 deletion in a mouse model led to early puberty onset in female mice. We found that Mkrn3 deletion increased the number of dendritic spines in the arcuate nucleus but did not alter the morphology of GnRH neurons during postnatal development. In addition, we identified neurokinin B (NKB) as an Mkrn3 target. Using proteomics, we identified insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) as another target of MKRN3. Interactome analysis revealed that IGF2BP1 interacted with MKRN3, along with several members of the polyadenylate-binding protein family. Our data show that one of the mechanisms by which MKRN3 inhibits pubertal initiation is through regulation of prepubertal hypothalamic development and plasticity, as well as through effects on NKB and IGF2BP1.
Lydie Naulé, Alessandra Mancini, Sidney A. Pereira, Brandon M. Gassaway, John R. Lydeard, John C. Magnotto, Han Kyeol Kim, Joy Liang, Cynara Matos, Steven P. Gygi, Florian T. Merkle, Rona S. Carroll, Ana Paula Abreu, Ursula B. Kaiser
Cerebrovasculature is critical in maintaining brain homeostasis; its dysregulation often leads to vascular cognitive impairment and dementia (VCID) during aging. VCID is the second most prevalent cause of dementia in the elderly, after Alzheimer’s disease (AD), with frequent cooccurrence of VCID and AD. While multiple factors are involved in the pathogenesis of AD and VCID, APOE4 increases the risk for both diseases. A major apolipoprotein E (apoE) receptor, the low-density lipoprotein receptor-related protein 1 (LRP1), is abundantly expressed in vascular mural cells (pericytes and smooth muscle cells). Here, we investigated how deficiency of vascular mural cell LRP1 affects the cerebrovascular system and cognitive performance using vascular mural cell–specific Lrp1-KO mice (smLrp1–/–) in a human APOE3 or APOE4 background. We found that spatial memory was impaired in the 13- to 16-month-old APOE4 smLrp1–/– mice but not in the APOE3 smLrp1–/– mice, compared with their respective littermate control mice. These disruptions in the APOE4 smLrp1–/– mice were accompanied with excess paravascular glial activation and reduced cerebrovascular collagen IV. In addition, blood-brain barrier (BBB) integrity was disrupted in the APOE4 smLrp1–/– mice. Together, our results suggest that vascular mural cell LRP1 modulates cerebrovasculature integrity and function in an APOE genotype–dependent manner.
Hiroshi Oue, Yu Yamazaki, Wenhui Qiao, Chen Yuanxin, Yingxue Ren, Aishe Kurti, Francis Shue, Tammee M. Parsons, Ralph B. Perkerson, Keiji Kawatani, Ni Wang, Skylar C. Starling, Bhaskar Roy, Ioana-Emilia Mosneag, Tomonori Aikawa, Marie-Louise Holm, Chia-Chen Liu, Yasuteru Inoue, Patrick M. Sullivan, Yan W. Asmann, Betty Y.S. Kim, Guojun Bu, Takahisa Kanekiyo
GM3 synthase deficiency (GM3SD) is an infantile-onset epileptic encephalopathy syndrome caused by biallelic loss-of-function mutations in ST3GAL5. Loss of ST3GAL5 activity in humans results in systemic ganglioside deficiency and severe neurological impairment. No disease-modifying treatment is currently available. Certain recombinant adeno-associated viruses (rAAVs) are capable of crossing the blood-brain barrier to induce widespread, long-term gene expression in the central nervous system (CNS), and represent a promising therapeutic strategy. Here, we show that a first-generation rAAV-ST3GAL5 replacement vector employing a ubiquitous promoter restored tissue ST3GAL5 expression and normalized cerebral gangliosides in patient-derived iPSC neurons and brain tissue from St3gal5 knock-out mice, but caused fatal hepatotoxicity when administered systemically. In contrast, a second-generation vector optimized for CNS-restricted ST3GAL5 expression, administered by either intracerebroventricular or intravenous route at postnatal day 1, allowed for safe and effective rescue of lethality and behavior impairment in symptomatic GM3SD mice up to a year. These results support further clinical development of ST3GAL5 gene therapy.
Huiya Yang, Robert Brown, Dan Wang, Kevin A. Strauss, Guangping Gao
Sorbitol dehydrogenase (SORD) deficiency has been identified as the most frequent autosomal recessive form of hereditary neuropathy. Loss of SORD causes high sorbitol levels in tissues due to the inability to convert sorbitol to fructose in the two-step polyol pathway, leading to degenerative neuropathy. The underlying mechanisms of sorbitol-induced degeneration have not been fully elucidated, and no current FDA-approved therapeutic options are available to reduce sorbitol levels in the nervous system. Here, in a Drosophila model of SORD deficiency, we showed synaptic degeneration in the brain, neurotransmission defect, locomotor impairment, and structural abnormalities in the neuromuscular junctions. In addition, we found reduced ATP production in the brain and reactive oxygen species accumulation in the central nervous system (CNS) and muscle, indicating mitochondrial dysfunction. Applied Therapeutics, Inc has developed a CNS-penetrant next-generation aldose reductase inhibitor (ARI), AT-007 (govorestat), which inhibits the conversion of glucose to sorbitol. AT-007 significantly reduced sorbitol levels in patient-derived fibroblasts, iPSC-derived motor neurons, and Drosophila brains. AT-007 feeding in Sord-deficient Drosophila mitigated synaptic degeneration and significantly improved synaptic transduction, locomotor activity, and mitochondrial function. Moreover, AT-007 treatment significantly reduced ROS accumulation in Drosophila CNS, muscle, and patient-derived fibroblasts. These findings uncover the molecular and cellular pathophysiology of SORD neuropathy and provide a potential treatment strategy for patients with SORD deficiency.
Yi Zhu, Amanda G. Lobato, Adriana P. Rebelo, Tijana Canic, Natalie Ortiz Vega, Xianzun Tao, Sheyum Syed, Christopher Yanick, Mario Saporta, Michael Shy, Riccardo Perfetti, Shoshana Shendelman, Stephan L. Zuchner, R. Grace Zhai
The prevalence of obesity and type 2 diabetes is growing at an alarming rate, including among pregnant women. Low-calorie sweeteners (LCS) have increasingly been used as an alternative to sugar to deliver a sweet taste without the excessive caloric load. However, there is little evidence regarding their biological effects, particularly during development. Here, we used a mouse model of maternal LCS consumption to explore the impact of perinatal LCS exposure on the development of neural systems involved in metabolic regulations. We report that adult male, but not female, offspring from both aspartame- and rebaudioside A-exposed dams displayed increased adiposity and developed glucose intolerance. Moreover, maternal LCS consumption reorganized hypothalamic melanocortin circuits and disrupted parasympathetic innervation of pancreatic islets in male offspring. We then identified phenylacetylglycine (PAG) as a unique metabolite that is upregulated in the milk of LCS-fed dams and the serum of their pups. Furthermore, maternal PAG treatment recapitulates some of the key metabolic and neurodevelopmental abnormalities associated with maternal LCS consumption. Together, our data indicate that maternal LCS consumption has enduring consequences on the offspring's metabolism and neural development and that these effects are likely to be mediated through the gut microbial co-metabolite PAG.
Soyoung Park, Amine M. Belfoul, Marialetizia Rastelli, Alice Jang, Magali Monnoye, Hosung Bae, Anna Kamitakahara, Patrick Giavalisco, Shan Sun, Pierre-Yves Barelle, Jasmine Plows, Cholsoon Jang, Anthony Fodor, Michael I. Goran, Sebastien G. Bouret
Gain-of-function mutations in the housekeeping gene GARS1, which lead to the expression of toxic versions of glycyl-tRNA synthetase (GlyRS), cause the selective motor and sensory pathology characterising Charcot-Marie-Tooth disease (CMT). Aberrant interactions between GlyRS mutants and different proteins, including neurotrophin receptor TrkB, underlie CMT type 2D (CMT2D); however, our pathomechanistic understanding of this untreatable peripheral neuropathy remains incomplete. Through intravital imaging of the sciatic nerve, we show that CMT2D mice display early and persistent disturbances in axonal transport of neurotrophin-containing signalling endosomes in vivo. We discovered that BDNF-TrkB impairments correlate with transport disruption and overall CMT2D neuropathology, and that inhibition of this pathway at the nerve-muscle interface perturbs endosome transport in wild-type axons. Accordingly, supplementation of muscles with BDNF, but not other neurotrophins, completely restores physiological axonal transport in neuropathic mice. Together, these findings suggest that selectively targeting muscles with BDNF-boosting therapies could represent a viable therapeutic strategy for CMT2D.
James N. Sleigh, David Villarroel-Campos, Sunaina Surana, Tahmina Wickenden, Yao Tong, Rebecca L. Simkin, Jose Norberto S. Vargas, Elena R. Rhymes, Andrew P. Tosolini, Steven J. West, Qian Zhang, Xiang-Lei Yang, Giampietro Schiavo
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