Progress in neurodegenerative disease research is hampered by the lack of biomarkers of neuronal dysfunction. We here identified a class of cerebrospinal fluid–based (CSF-based) kinetic biomarkers that reflect altered neuronal transport of protein cargo, a common feature of neurodegeneration. After a pulse administration of heavy water (2H2O), distinct, newly synthesized 2H-labeled neuronal proteins were transported to nerve terminals and secreted, and then appeared in CSF. In 3 mouse models of neurodegeneration, distinct 2H-cargo proteins displayed delayed appearance and disappearance kinetics in the CSF, suggestive of aberrant transport kinetics. Microtubule-modulating pharmacotherapy normalized CSF-based kinetics of affected 2H-cargo proteins and ameliorated neurodegenerative symptoms in mice. After 2H2O labeling, similar neuronal transport deficits were observed in CSF of patients with Parkinson’s disease (PD) compared with non-PD control subjects, which indicates that these biomarkers are translatable and relevant to human disease. Measurement of transport kinetics may provide a sensitive method to monitor progression of neurodegeneration and treatment effects.
Patrizia Fanara, Po-Yin A. Wong, Kristofor H. Husted, Shanshan Liu, Victoria M. Liu, Lori A. Kohlstaedt, Timothy Riiff, Joan C. Protasio, Drina Boban, Salena Killion, Maudi Killian, Lorrie Epling, Elisabeth Sinclair, Julia Peterson, Richard W. Price, Deborah E. Cabin, Robert L. Nussbaum, Jörg Brühmann, Roland Brandt, Chadwick W. Christine, Michael J. Aminoff, Marc K. Hellerstein
Amyotrophic lateral sclerosis (ALS) is a progressive disease associated with neuronal cell death that is thought to involve aberrant immune responses. Here we investigated the role of innate immunity in a mouse model of ALS. We found that inflammatory monocytes were activated and that their progressive recruitment to the spinal cord, but not brain, correlated with neuronal loss. We also found a decrease in resident microglia in the spinal cord with disease progression. Prior to disease onset, splenic Ly6Chi monocytes expressed a polarized macrophage phenotype (M1 signature), which included increased levels of chemokine receptor CCR2. As disease onset neared, microglia expressed increased CCL2 and other chemotaxis-associated molecules, which led to the recruitment of monocytes to the CNS by spinal cord–derived microglia. Treatment with anti-Ly6C mAb modulated the Ly6Chi monocyte cytokine profile, reduced monocyte recruitment to the spinal cord, diminished neuronal loss, and extended survival. In humans with ALS, the analogous monocytes (CD14+CD16–) exhibited an ALS-specific microRNA inflammatory signature similar to that observed in the ALS mouse model, linking the animal model and the human disease. Thus, the profile of monocytes in ALS patients may serve as a biomarker for disease stage or progression. Our results suggest that recruitment of inflammatory monocytes plays an important role in disease progression and that modulation of these cells is a potential therapeutic approach.
Oleg Butovsky, Shafiuddin Siddiqui, Galina Gabriely, Amanda J. Lanser, Ben Dake, Gopal Murugaiyan, Camille E. Doykan, Pauline M. Wu, Reddy R. Gali, Lakshmanan K. Iyer, Robert Lawson, James Berry, Anna M. Krichevsky, Merit E. Cudkowicz, Howard L. Weiner
The second-largest cause of X-linked mental retardation is a deficiency in creatine transporter (CRT; encoded by SLC6A8), which leads to speech and language disorders with severe cognitive impairment. This syndrome, caused by the absence of creatine in the brain, is currently untreatable because CRT is required for creatine entry into brain cells. Here, we developed a brain-specific Slc6a8 knockout mouse (Slc6a8–/y) as an animal model of human CRT deficiency in order to explore potential therapies for this syndrome. The phenotype of the Slc6a8–/y mouse was comparable to that of human patients. We successfully treated the Slc6a8–/y mice with the creatine analog cyclocreatine. Brain cyclocreatine and cyclocreatine phosphate were detected after 9 weeks of cyclocreatine treatment in Slc6a8–/y mice, in contrast to the same mice treated with creatine or placebo. Cyclocreatine-treated Slc6a8–/y mice also exhibited a profound improvement in cognitive abilities, as seen with novel object recognition as well as spatial learning and memory tests. Thus, cyclocreatine appears promising as a potential therapy for CRT deficiency.
Yuko Kurosawa, Ton J. DeGrauw, Diana M. Lindquist, Victor M. Blanco, Gail J. Pyne-Geithman, Takiko Daikoku, James B. Chambers, Stephen C. Benoit, Joseph F. Clark
Pain and depression are frequently comorbid disorders, but the mechanism underlying this association is unknown. Here, we report that brain indoleamine 2,3-dioxygenase 1 (IDO1), a rate-limiting enzyme in tryptophan metabolism, plays a key role in this comorbidity. We found that chronic pain in rats induced depressive behavior and IDO1 upregulation in the bilateral hippocampus. Upregulation of IDO1 resulted in the increased kynurenine/tryptophan ratio and decreased serotonin/tryptophan ratio in the bilateral hippocampus. We observed elevated plasma IDO activity in patients with both pain and depression, as well as in rats with anhedonia induced by chronic social stress. Intra-hippocampal administration of IL-6 in rats, in addition to in vitro experiments, demonstrated that IL-6 induces IDO1 expression through the JAK/STAT pathway. Further, either Ido1 gene knockout or pharmacological inhibition of hippocampal IDO1 activity attenuated both nociceptive and depressive behavior. These results reveal an IDO1-mediated regulatory mechanism underlying the comorbidity of pain and depression and suggest a new strategy for the concurrent treatment of both conditions via modulation of brain IDO1 activity.
Hyangin Kim, Lucy Chen, Grewo Lim, Backil Sung, Shuxing Wang, Michael F. McCabe, Gabriel Rusanescu, Liling Yang, Yinghong Tian, Jianren Mao
Congenital myasthenic syndromes (CMSs) are neuromuscular disorders that can be caused by defects in acetylcholine receptor (AChR) function. Disease-associated point mutants can reveal the unsuspected functional significance of mutated residues. We identified two pathogenic mutations in the extracellular domain of the AChR α subunit (AChRα) in a patient with myasthenic symptoms since birth: a V188M mutation in the C-loop and a heteroallelic G74C mutation in the main immunogenic region. The G74C mutation markedly reduced surface AChR expression in cultured cells, whereas the V188M mutant was expressed robustly but had severely impaired kinetics. Single-channel patch-clamp analysis indicated that V188M markedly decreased the apparent AChR channel opening rate and gating efficiency. Mutant cycle analysis of energetic coupling among conserved residues within or dispersed around the AChRα C-loop revealed that V188 is functionally linked to Y190 in the C-loop and to D200 in β-strand 10, which connects to the M1 transmembrane domain. Furthermore, V188M weakens inter-residue coupling of K145 in β-strand 7 with Y190 and with D200. Cumulatively, these results indicate that V188 of AChRα is part of an interdependent tetrad that contributes to rearrangement of the C-loop during the initial coupling of agonist binding to channel gating.
Xin-Ming Shen, Joan M. Brengman, Steven M. Sine, Andrew G. Engel
An emerging theory of schizophrenia postulates that hypofunction of adenosine signaling may contribute to its pathophysiology. This study was designed to test the “adenosine hypothesis” of schizophrenia and to evaluate focal adenosine-based strategies for therapy. We found that augmentation of adenosine by pharmacologic inhibition of adenosine kinase (ADK), the key enzyme of adenosine clearance, exerted antipsychotic-like activity in mice. Further, overexpression of ADK in transgenic mice was associated with attentional impairments linked to schizophrenia. We observed that the striatal adenosine A2A receptor links adenosine tone and psychomotor response to amphetamine, an indicator of dopaminergic signaling. Finally, intrastriatal implants of engineered adenosine-releasing cells restored the locomotor response to amphetamine in mice overexpressing ADK, whereas the same grafts placed proximal to the hippocampus of transgenic mice reversed their working memory deficit. This functional double dissociation between striatal and hippocampal adenosine demonstrated in Adk transgenic mice highlights the independent contributions of these two interconnected brain regions in the pathophysiology of schizophrenia and thus provides the rationale for developing local adenosine augmentation therapies for the treatment of schizophrenia.
Hai-Ying Shen, Philipp Singer, Nikki Lytle, Catherine J. Wei, Jing-Quan Lan, Rebecca L. Williams-Karnesky, Jiang-Fan Chen, Benjamin K. Yee, Detlev Boison
Stressful events during early childhood can have a profound lifelong influence on emotional and cognitive behaviors. However, the mechanisms by which stress affects neonatal brain circuit formation are poorly understood. Here, we show that neonatal social isolation disrupts molecular, cellular, and circuit developmental processes, leading to behavioral dysfunction. Neonatal isolation prevented long-term potentiation and experience-dependent synaptic trafficking of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors normally occurring during circuit formation in the rodent barrel cortex. This inhibition of AMPA receptor trafficking was mediated by an increase of the stress glucocorticoid hormone and was associated with reduced calcium/calmodulin-dependent protein kinase type II (CaMKII) signaling, resulting in attenuated whisker sensitivity at the cortex. These effects led to defects in whisker-dependent behavior in juvenile animals. These results indicate that neonatal social isolation alters neuronal plasticity mechanisms and perturbs the initial establishment of a normal cortical circuit, which potentially explains the long-lasting behavioral effects of neonatal stress.
Tomoyuki Miyazaki, Kenkichi Takase, Waki Nakajima, Hirobumi Tada, Daisuke Ohya, Akane Sano, Takahisa Goto, Hajime Hirase, Roberto Malinow, Takuya Takahashi
In inflammatory CNS conditions such as multiple sclerosis (MS), current options to treat clinical relapse are limited, and more selective agents are needed. Disruption of the blood-brain barrier (BBB) is an early feature of lesion formation that correlates with clinical exacerbation, leading to edema, excitotoxicity, and entry of serum proteins and inflammatory cells. Here, we identify astrocytic expression of VEGF-A as a key driver of BBB permeability in mice. Inactivation of astrocytic Vegfa expression reduced BBB breakdown, decreased lymphocyte infiltration and neuropathology in inflammatory and demyelinating lesions, and reduced paralysis in a mouse model of MS. Knockdown studies in CNS endothelium indicated activation of the downstream effector eNOS as the principal mechanism underlying the effects of VEGF-A on the BBB. Systemic administration of the selective eNOS inhibitor cavtratin in mice abrogated VEGF-A–induced BBB disruption and pathology and protected against neurologic deficit in the MS model system. Collectively, these data identify blockade of VEGF-A signaling as a protective strategy to treat inflammatory CNS disease.
Azeb Tadesse Argaw, Linnea Asp, Jingya Zhang, Kristina Navrazhina, Trinh Pham, John N. Mariani, Sean Mahase, Dipankar J. Dutta, Jeremy Seto, Elisabeth G. Kramer, Napoleone Ferrara, Michael V. Sofroniew, Gareth R. John
Leigh syndrome (LS) is a subacute necrotizing encephalomyelopathy with gliosis in several brain regions that usually results in infantile death. Loss of murine Ndufs4, which encodes NADH dehydrogenase (ubiquinone) iron-sulfur protein 4, results in compromised activity of mitochondrial complex I as well as progressive neurodegenerative and behavioral changes that resemble LS. Here, we report the development of breathing abnormalities in a murine model of LS. Magnetic resonance imaging revealed hyperintense bilateral lesions in the dorsal brain stem vestibular nucleus (VN) and cerebellum of severely affected mice. The mutant mice manifested a progressive increase in apnea and had aberrant responses to hypoxia. Electrophysiological recordings within the ventral brain stem pre-Bötzinger respiratory complex were also abnormal. Selective inactivation of Ndufs4 in the VN, one of the principle sites of gliosis, also led to breathing abnormalities and premature death. Conversely, Ndufs4 restoration in the VN corrected breathing deficits and prolonged the life span of knockout mice. These data demonstrate that mitochondrial dysfunction within the VN results in aberrant regulation of respiration and contributes to the lethality of Ndufs4-knockout mice.
Albert Quintana, Sebastien Zanella, Henner Koch, Shane E. Kruse, Donghoon Lee, Jan M. Ramirez, Richard D. Palmiter
Glyoxalase 1 (Glo1) expression has previously been associated with anxiety in mice; however, its role in anxiety is controversial, and the underlying mechanism is unknown. Here, we demonstrate that GLO1 increases anxiety by reducing levels of methylglyoxal (MG), a GABAA receptor agonist. Mice overexpressing Glo1 on a Tg bacterial artificial chromosome displayed increased anxiety-like behavior and reduced brain MG concentrations. Treatment with low doses of MG reduced anxiety-like behavior, while higher doses caused locomotor depression, ataxia, and hypothermia, which are characteristic effects of GABAA receptor activation. Consistent with these data, we found that physiological concentrations of MG selectively activated GABAA receptors in primary neurons. These data indicate that GLO1 increases anxiety by reducing levels of MG, thereby decreasing GABAA receptor activation. More broadly, our findings potentially link metabolic state, neuronal inhibitory tone, and behavior. Finally, we demonstrated that pharmacological inhibition of GLO1 reduced anxiety, suggesting that GLO1 is a possible target for the treatment of anxiety disorders.
Margaret G. Distler, Leigh D. Plant, Greta Sokoloff, Andrew J. Hawk, Ivy Aneas, Gerald E. Wuenschell, John Termini, Stephen C. Meredith, Marcelo A. Nobrega, Abraham A. Palmer