Hypocretin-2 Saporin Lesions of the Ventrolateral Periaquaductal Gray (vlPAG) Increase REM Sleep in Hypocretin Knockout Mice

Ten years ago the sleep disorder narcolepsy was linked to the neuropeptide hypocretin (HCRT), also known as orexin. This disorder is characterized by excessive day time sleepiness, inappropriate triggering of rapid-eye movement (REM) sleep and cataplexy, which is a sudden loss of muscle tone during waking. It is still not known how HCRT regulates REM sleep or muscle tone since HCRT neurons are localized only in the lateral hypothalamus while REM sleep and muscle atonia are generated from the brainstem. To identify a potential neuronal circuit, the neurotoxin hypocretin-2-saporin (HCRT2-SAP) was used to lesion neurons in the ventral lateral periaquaductal gray (vlPAG). The first experiment utilized hypocretin knock-out (HCRT-ko) mice with the expectation that deletion of both HCRT and its target neurons would exacerbate narcoleptic symptoms. Indeed, HCRT-ko mice (n = 8) given the neurotoxin HCRT2-SAP (16.5 ng/23nl/sec each side) in the vlPAG had levels of REM sleep and sleep fragmentation that were considerably higher compared to HCRT-ko given saline (+39%; n = 7) or wildtype mice (+177%; n = 9). However, cataplexy attacks did not increase, nor were levels of wake or non-REM sleep changed. Experiment 2 determined the effects in mice where HCRT was present but the downstream target neurons in the vlPAG were deleted by the neurotoxin. This experiment utilized an FVB-transgenic strain of mice where eGFP identifies GABA neurons. We verified this and also determined that eGFP neurons were immunopositive for the HCRT-2 receptor. vlPAG lesions in these mice increased REM sleep (+79% versus saline controls) and it was significantly correlated (r = 0.89) with loss of eGFP neurons. These results identify the vlPAG as one site that loses its inhibitory control over REM sleep, but does not cause cataplexy, as a result of hypocretin deficiency.     

Adipose tissue-derived stem cells rescue Purkinje neurons and alleviate inflammatory responses in Niemann-Pick disease type C mice

Adult stem cells offer special therapeutic prospects because they can be isolated for autologous transplantation, expanded ex vivo, and differentiated into various cell types. We previously reported that bone marrow-derived mesenchymal stem cells improve neurological deficits in neurodegenerative disease animal models. However, the efficacy of adipose tissue-derived stem cells (ADSCs) transplantation in similar models remains unknown. Herein, we demonstrate that ADSCs, when transplanted into Niemann-Pick disease type C (NP-C) mouse cerebellum, elicit rescue of Purkinje neurons and restoration of motor coordination together with alleviation of inflammatory responses as verified by immunohistochemistry and real-time PCR using glial fibrillary acidic protein (GFAP), F4/80, IL-1β, IL-6, and TNF-α. Most importantly, ADSCs enhance electrically active Purkinje neurons with functional synaptic formation after transplantation in NP-C disease model mice. This report demonstrates for the first time that ADSCs can rescue imperiled Purkinje neurons and alleviate the inflammatory response in NP-C disease model mice, thereby signifying the therapeutic potential of ADSCs for neurodegenerative diseases.     

Chemical conversion of human and mouse fibroblasts into motor neurons

Transplantation of motor neurons can provide long-term functional benefits in animal models of neurodegenerative motor neuron diseases such as amyotrophic lateral sclerosis and traumatic spinal cord injury. Although embryonic stem cells can differentiate into motor neurons, alternative sources of motor neurons may be controllable for disease modeling and transplantation. Here, we show that human and mouse fibroblasts can be efficiently and directly converted into motor neurons by a cocktail of five small molecules, without the involvement of the neural progenitor stage. The chemically-induced motor neurons display the distinct neuronal morphology and express motor neuron markers. Interestingly, when the same chemical compounds were soaked in beads and implanted in the hypodermis of the back skins of mice, surrounding cells begin to express motor neuron markers, indicating in vivo motor neuron reprogramming. Taken together, we provide an efficient approach for chemically converting human and mouse fibroblasts into motor neurons suitable for cell replacement therapy and neurodegenerative disease modeling.     

Heat shock protein 60 affects behavioral improvement in a rat model of Parkinson’s disease grafted with human umbilical cord mesenchymal stem cell-derived dopaminergic-like neurons

Parkinson’s disease (PD) is a neurodegenerative disorder that is caused by a loss of dopaminergic (DAergic) neurons in mesencephalic substantia nigra (SN). Human umbilical cord mesenchymal stem cells (hUC-MSCs) are capable of self-renewal and differentiation into multiple cell lineages, including DAergic neurons. Thus, hUC-MSCs could be a promising alternative to compensate for the loss of DAergic neurons in PD. In the current study, hUC-MSCs and hUC-MSCs-derived DAergic-like neurons were transplanted into the striatum and SN of a rat model of PD that is induced by 6-hydroxydopamine (6-OHDA). We evaluated their therapeutic effects on improving rotation behavior in the rat and on modulating the level of heat shock protein 60 (Hsp60) expression in the brain. After transplantation, an amelioration of rotation behavior was observed in rats that underwent cell grafting, and hUC-MSCs-derived DAergic-like neurons were superior to hUC-MSCs at inducing behavioral improvement. Western blot and immunohistochemistry analysis indicated significantly elevated levels of Hsp60 in cell-grafted rats compared to 6-OHDA-lesioned (PD) rats. These results demonstrate that hUC-MSCs-based cell transplantation is potential therapeutic treatment for PD, and hUC-MSCs-derived DAergic-like neurons appear to be favorable candidates for cell replacement therapy in PD. Finally, Hsp60 could be involved in a mechanism of behavioral recovery.     

Stem cell‐based therapy for Huntington's disease

Huntington's disease (HD) is a late‐onset neurodegenerative disease characterized by a progressive loss of medium spiny neurons in the basal ganglia. The development of stem cell‐based therapies for HD aims to replace lost neurons and/or to prevent cell death. This review will discuss pre‐clinical studies which have utilized stem or progenitor cells for transplantation therapy using HD animal models. In several studies, neural stem and progenitor cells used as allotransplants and xenografts have been shown to be capable of surviving transplantation and differentiating into mature GABAergic neurons, resulting in behavioral improvements. Beneficial effects have also been reported for transplantation of stem cells derived from non‐neural tissue, for example, mesenchymal‐ and adipose‐derived stem cells, which have mainly been attributed to their secretion of growth and neurotrophic factors. Finally, we review studies using stem cells genetically engineered to over‐express defined neurotrophic factors. While these studies prove the potential of stem cells for transplantation therapy in HD, it also becomes clear that technical and ethical issues regarding the availability of stem cells must be solved before human trials can be conducted. J. Cell. Biochem. 114: 754–763, 2013. © 2012 Wiley Periodicals, Inc.     

表达神经营养因子Neurturin的骨髓间充质干细胞的构建及体外活性检测

研究神经营养因子Neurturin(NTN)在由于神经元损伤而造成的神经退行性疾病中对神经元的保护和修复作用。利用重组腺病毒载体将NTN基因转入恒河猴骨髓间充质干细胞(rMSC),通过RT-PCR、IF及Western blot方法检测NTN的转录和表达,并采用鸡胚背根神经节体外培养实验和胚胎大鼠中脑多巴胺能神经元存活实验对NTN进行体外活性检测。结果表明NTN在rMSC中稳定表达和分泌,并具有体外生物学活性,为由于神经元损伤造成的神经退行性疾病的干细胞移植治疗奠定了一定的基础。     

Control of cardiovascular variability during undisturbed wake-sleep behavior in hypocretin-deficient mice

The central neural mechanisms underlying differences in cardiovascular variability between wakefulness, non-rapid-eye-movement sleep (NREMS), and rapid-eye-movement sleep (REMS) remain poorly understood. These mechanisms may involve hypocretin (HCRT)/orexin signaling. HCRT signaling is linked to wake-sleep states, involved in central autonomic control, and impaired in narcoleptic patients. Thus, we investigated whether HCRT signaling plays a role in controlling cardiovascular variability during spontaneous behavior in HCRT-deficient mice. HCRT-ataxin3 transgenic mice lacking HCRT neurons (TG), knockout mice lacking HCRT peptides (KO), and wild-type controls (WT) were instrumented with electrodes for sleep recordings and a telemetric blood pressure transducer. Fluctuations of systolic blood pressure (SBP) and heart period (HP) during undisturbed wake-sleep behavior were analyzed with the sequence technique, cross-correlation functions, and coherent averaging of SBP surges. During NREMS, all mice had lower SBP variability, greater baroreflex contribution to HP control at low frequencies, and greater amplitude of the central autonomic and baroreflex changes in HP associated with SBP surges than during wakefulness. During REMS, all mice had higher SBP variability and depressed central autonomic and baroreflex HP controls relative to NREMS. HP variability during REMS was higher than during NREMS in WT only. TG and KO also had lower amplitude of the cardiac baroreflex response to SBP surges during REMS than WT. These results indicate that chronic lack of HCRT signaling may cause subtle alterations in the control of HP during spontaneous behavior. Conversely, the integrity of HCRT signaling is not necessary for the occurrence of physiological sleep-dependent changes in SBP variability.     

Circadian and homeostatic regulation of structural synaptic plasticity in hypocretin neurons

Neurons exhibit rhythmic activity that ultimately affects behavior such as sleep. In living zebrafish larvae, we used time-lapse two-photon imaging of the presynaptic marker synaptophysin in hypocretin/orexin (HCRT) neurons to determine the dynamics of synaptic modifications during the day and night. We observed circadian rhythmicity in synapse number in HCRT axons. This rhythm is regulated primarily by the circadian clock but is also affected by sleep deprivation. Furthermore, NPTX2, a protein implicated in AMPA receptor clustering, modulates circadian synaptic changes. In zebrafish, nptx2b is a rhythmic gene that is mostly expressed in hypothalamic and pineal gland cells. Arrhythmic transgenic nptx2b overexpression (hcrt:NPTX2b) increases synapse number and abolishes rhythmicity in HCRT axons. Finally, hcrt:NPTX2b fish are resistant to the sleep-promoting effects of melatonin. This behavioral effect is consistent with NPTX2b-mediated increased activity of HCRT circuitry. These data provide real-time in vivo evidence of circadian and homeostatic regulation of structural synaptic plasticity.     

Genetic ablation of orexin neurons in mice results in narcolepsy, hypophagia, and obesity

Orexins (hypocretins) are a pair of neuropeptides implicated in energy homeostasis and arousal. Recent reports suggest that loss of orexin-containing neurons occurs in human patients with narcolepsy. We generated transgenic mice in which orexin-containing neurons are ablated by orexinergic-specific expression of a truncated Machado-Joseph disease gene product (ataxin-3) with an expanded polyglutamine stretch. These mice showed a phenotype strikingly similar to human narcolepsy, including behavioral arrests, premature entry into rapid eye movement (REM) sleep, poorly consolidated sleep patterns, and a late-onset obesity, despite eating less than nontransgenic littermates. These results provide evidence that orexin-containing neurons play important roles in regulating vigilance states and energy homeostasis. Orexin/ataxin-3 mice provide a valuable model for studying the pathophysiology and treatment of narcolepsy.     

Narcolepsy,orexins and respiratory regulation

Narcolepsy is a debilitating sleep disorder characterized by excessive daytime sleepiness, cataplexy and intrusive rapid–eye movement sleep. Deficits in endogenous orexins are a major pathogenic component of the disease. This disorder is also associated with the gene marker HLADQB1*0602. Orexins as hypothalamic neuropeptides have multiple physiological functions, and their primary functions are regulation of the sleep–wake cycle and feeding. Evidence from animal studies using orexin knockout mice and focal microdialysis of an orexin receptor antagonist at the retrotrapezoid nucleus and medullary raphe in rats demonstrated that orexins also contribute to respiratory regulation in a vigilance state–dependent manner, as animals with orexin dysregulation have attenuated hypercapnic ventilatory responses predominantly in wakefulness. These findings are consistent with the notion that the activity of orexinergic neurons is higher during wake than sleep periods. Orexin neurons seem to be a pivotal link between conscious and unconscious brain functions in animals. The human model of hypocretin deficiency is patients with narcolepsy–cataplexy. In contrast to the findings suggested by animal studies, we found significant decreases in hypoxic responsiveness, but not in hypercapnic responsiveness, in narcoleptics, and further analysis indicated that decreased ventilatory responses to hypoxia in human narcolepsy–cataplexy is in relation to HLA-DQB1*0602 status, not hypocretin deficiency. This is confirmed by the fact that the hypoxic responsiveness was lower in HLA positive versus negative controls. Unlike in mice, hypocretin-1 is not a major factor contributing to depressed hypoxic responses in humans. Species differences may exist.

     

Reduced number of hypocretin neurons in human narcolepsy

Murine and canine narcolepsy can be caused by mutations of the hypocretin (Hcrt) (orexin) precursor or Hcrt receptor genes. In contrast to these animal models, most human narcolepsy is not familial, is discordant in identical twins, and has not been linked to mutations of the Hcrt system. Thus, the cause of human narcolepsy remains unknown. Here we show that human narcoleptics have an 85%-95% reduction in the number of Hcrt neurons. Melanin-concentrating hormone (MCH) neurons, which are intermixed with Hcrt cells in the normal brain, are not reduced in number, indicating that cell loss is relatively specific for Hcrt neurons. The presence of gliosis in the hypocretin cell region is consistent with a degenerative process being the cause of the Hcrt cell loss in narcolepsy.     

The nature of the cell cycle in neurons: Focus on a “non-canonical” pathway of DNA replication causally related to death

The mechanism whereby a reactivation of cell cycle in neurons causes cell death is beginning to be identified. In cellular models of Alzheimer's disease, activation of a non-canonical pathway of DNA replication contributes to neuronal death. This pathway involves the repair enzyme DNA polymerase-β, which is highly expressed in neurons of the Alzheimer's brain at early stages of the disease. Loading of DNA polymerase-β into the replication forks generates a death signal, which involves the tumor suppressor p53. The increasing knowledge of the main actors of the unscheduled DNA replication in neurons will pave the way for novel therapeutic interventions in Alzheimer's disease and other neurodegenerative disorders.     

Narcolepsy and familial advanced sleep-phase syndrome: molecular genetics of sleep disorders

Sleep disorders are very prevalent and represent an emerging worldwide epidemic. However, research into the molecular genetics of sleep disorders remains surprisingly one of the least active fields. Nevertheless, rapid progress is being made in several prototypical disorders, leading recently to the identification of the molecular pathways underlying narcolepsy and familial advanced sleep-phase syndrome. Since the first reports of spontaneous and induced loss-of-function mutations leading to hypocretin deficiency in human and animal models of narcolepsy, the role of this novel neurotransmission pathway in sleep and several other behaviors has gained extensive interest. Also, very recent studies using an animal model of familial advanced sleep-phase syndrome shed new light on the regulation of circadian rhythms.     

Genetically Engineered Mesenchymal Stem Cells as a Proposed Therapeutic for Huntington’s Disease

There is much interest in the use of mesenchymal stem cells/marrow stromal cells (MSC) to treat neurodegenerative disorders, in particular those that are fatal and difficult to treat, such as Huntington's disease. MSC present a promising tool for cell therapy and are currently being tested in FDA-approved phase I-III clinical trials for many disorders. In preclinical studies of neurodegenerative disorders, MSC have demonstrated efficacy, when used as delivery vehicles for neural growth factors. A number of investigators have examined the potential benefits of innate MSC-secreted trophic support and augmented growth factors to support injured neurons. These include overexpression of brain-derived neurotrophic factor and glial-derived neurotrophic factor, using genetically engineered MSC as a vehicle to deliver the cytokines directly into the microenvironment. Proposed regenerative approaches to neurological diseases using MSC include cell therapies in which cells are delivered via intracerebral or intrathecal injection. Upon transplantation, MSC in the brain promote endogenous neuronal growth, encourage synaptic connection from damaged neurons, decrease apoptosis, reduce levels of free radicals, and regulate inflammation. These abilities are primarily modulated through paracrine actions. Clinical trials for MSC injection into the central nervous system to treat amyotrophic lateral sclerosis, traumatic brain injury, and stroke are currently ongoing. The current data in support of applying MSC-based cellular therapies to the treatment of Huntington's disease is discussed.     

Microtubule-stabilizing agents as potential therapeutics for neurodegenerative disease

Microtubules (MTs), cytoskeletal elements found in all mammalian cells, play a significant role in cell structure and in cell division. They are especially critical in the proper functioning of post-mitotic central nervous system neurons, where MTs serve as the structures on which key cellular constituents are trafficked in axonal projections. MTs are stabilized in axons by the MT-associated protein tau, and in several neurodegenerative diseases, including Alzheimer’s disease, frontotemporal lobar degeneration, and Parkinson’s disease, tau function appears to be compromised due to the protein dissociating from MTs and depositing into insoluble inclusions referred to as neurofibrillary tangles. This loss of tau function is believed to result in alterations of MT structure and function, resulting in aberrant axonal transport that likely contributes to the neurodegenerative process. There is also evidence of axonal transport deficiencies in other neurodegenerative diseases, including amyotrophic lateral sclerosis and Huntington’s disease, which may result, at least in part, from MT alterations. Accordingly, a possible therapeutic strategy for such neurodegenerative conditions is to treat with MT-stabilizing agents, such as those that have been used in the treatment of cancer. Here, we review evidence of axonal transport and MT deficiencies in a number of neurodegenerative diseases, and summarize the various classes of known MT-stabilizing agents. Finally, we highlight the growing evidence that small molecule MT-stabilizing agents provide benefit in animal models of neurodegenerative disease and discuss the desired features of such molecules for the treatment of these central nervous system disorders.     

How hypocretin agonists may improve the quality of wake in narcolepsy

Excessive daytime sleepiness (EDS) is a complex symptom characterized by a strong urge to sleep during daytime accompanied by problems such as attention deficits, anxiety, and lower cognitive performance. The efficacy of treatments for EDS is determined by their ability to decrease sleepiness, and less attention has been given to the effects these compounds have on the quality of the wake itself. Hypocretin (HCRT; orexin) signalling is implicated in narcolepsy, and hypocretin receptor 2 (HCRTR2) agonists are in clinical trials for treating EDS in narcolepsy. Here, we review preclinical research to determine how HCRTR2 agonists may affect attention and anxiety compared with other EDS treatment strategies. We conclude that such compounds may improve not only the quantity but also the quality of wake, and we hope that they will create opportunities for more nuanced treatment strategies in narcolepsy.     

Sertoli cell transplants: their use in the treatment of neurodegenerative disease

The efficacy of treating neurodegenerative diseases with the transplantation of fetal tissue has been demonstrated in animal models of Parkinson's disease, Huntington's disease and stroke. In the clinical setting, neural transplantation as a treatment for patients with Parkinson's disease has shown promising results. However, for this treatment method to be effective neuronal survival needs to be improved through either trophic support or localized immunoprotection. Co-transplanting Sertoli cells, which express many nutritive, regulatory, trophic and immunosuppressive factors, with fetal neural cells could provide both of these requirements. Such a strategy could enhance the recovery benefits associated with transplantation and decrease the need for, and the risks associated with, long-term systemic immunosuppression.     

Regenerative medicine for Parkinson’s disease using differentiated nerve cells derived from human buccal fat pad stem cells

The purpose of this study was to evaluate the utility of human adipose stem cells derived from the buccal fat pad (hBFP-ASCs) for nerve regeneration. Parkinson’s disease (PD) is a neurodegenerative disorder characterized by progressive death of dopaminergic neurons. PD is a candidate disease for cell replacement therapy because it has no fundamental therapeutic methods. We examined the properties of neural-related cells induced from hBFP-ASCs as a cell source for PD treatment. hBFP-ASCs were cultured in neurogenic differentiation medium for about 2 weeks. After the morphology of hBFP-ASCs changed to neural-like cells, the medium was replaced with neural maintenance medium. Cells differentiated from hBFP-ASCs showed neuron-like structures and expressed neuron markers (β3-tubulin, neurofilament 200, and microtubule-associated protein 2), an astrocyte marker (glial fibrillary acidic protein), or dopaminergic neuron-related marker (tyrosine hydroxylase). Induced neural cells were transplanted into a 6-hydroxydopamine (6-OHDA)-lesioned rat hemi-parkinsonian model. At 4 weeks after transplantation, 6-OHDA-lesioned rats were subjected to apomorphine-induced rotation analysis. The transplanted cells survived in the brain of rats as dopaminergic neural cells. No tumor formation was found after cell transplantation. We demonstrated differentiation of hBFP-ASCs into neural cells, and that transplantation of these neural cells improved the symptoms of model rats. Our results suggest that neurons differentiated from hBFP-ASCs would be applicable to cell replacement therapy of PD.