Inhibition of polyglutamine aggregation in R6/2 HD brain slices-complex dose-response profiles.

Huntington's disease (HD) is a late onset neurodegenerative disorder caused by a CAG/polyglutamine (polyQ) repeat expansion. PolyQ aggregates can be detected in the nuclei and processes of neurons in HD patients and mouse models prior to the onset of symptoms. The misfolding and aggregation pathway is an important therapeutic target. To better test the efficacy of aggregation inhibitors, we have developed an organotypic slice culture system. We show here that the formation of polyQ aggregates in hippocampal slices established from the R6/2 mouse follows the same prescribed sequence as occurs in vivo. Using this assay, we show that Congo red and chrysamine G can modulate aggregate formation, but show complex dose-response curves. Oral administration of creatine has been shown to delay the onset of all aspects of the phenotype and neuropathology in R6/2 mice. We show here that creatine can similarly inhibit aggregate formation in the slice culture assay.     

Small-molecule inhibitors of bone morphogenic protein and activin/nodal signals promote highly efficient neural induction from human pluripotent stem cells

The balance of bone morphogenic protein (BMP), transforming growth factor-β (TGFβ)/activin/nodal, and Wnt signals regulates the early lineage segregation of human embryonic stem cells (ESCs). Here we demonstrate that a combination of small-molecule inhibitors of BMP (Dorsomorphin) and TGFβ/activin/nodal (SB431542) signals promotes highly efficient neural induction from both human ESCs and induced pluripotent stem cells (iPSCs). The combination of small molecules had effects on both cell survival and purity of neural differentiation, under conditions of stromal (PA6) cell coculture and feeder-free floating aggregation culture, for all seven pluripotent stem cell lines that we studied, including three ESC and four iPSC lines. Small molecule compounds are stable and cost effective, so our findings provide a promising strategy for controlled production of neurons in regenerative medicine.     

Inhibition of Polyglutamine Aggregation in R6/2 HD Brain Slices—Complex Dose–Response Profiles

Huntington's disease (HD) is a late onset neurodegenerative disorder caused by a CAG/polyglutamine (polyQ) repeat expansion. PolyQ aggregates can be detected in the nuclei and processes of neurons in HD patients and mouse models prior to the onset of symptoms. The misfolding and aggregation pathway is an important therapeutic target. To better test the efficacy of aggregation inhibitors, we have developed an organotypic slice culture system. We show here that the formation of polyQ aggregates in hippocampal slices established from the R6/2 mouse follows the same prescribed sequence as occurs in vivo. Using this assay, we show that Congo red and chrysamine G can modulate aggregate formation, but show complex dose–response curves. Oral administration of creatine has been shown to delay the onset of all aspects of the phenotype and neuropathology in R6/2 mice. We show here that creatine can similarly inhibit aggregate formation in the slice culture assay.     

Experimental Models for Identifying Modifiers of Polyglutamine-Induced Aggregation and Neurodegeneration

Huntington’s disease (HD) typifies a class of inherited neurodegenerative disorders in which a CAG expansion in a single gene leads to an extended polyglutamine tract and misfolding of the expressed protein, driving cumulative neural dysfunction and degeneration. HD is invariably fatal with symptoms that include progressive neuropsychiatric and cognitive impairments, and eventual motor disability. No curative therapies yet exist for HD and related polyglutamine diseases; therefore, substantial efforts have been made in the drug discovery field to identify potential drug and drug target candidates for disease-modifying treatment. In this context, we review here a range of early-stage screening approaches based in in vitro, cellular, and invertebrate models to identify pharmacological and genetic modifiers of polyglutamine aggregation and induced neurodegeneration. In addition, emerging technologies, including high-content analysis, three-dimensional culture models, and induced pluripotent stem cells are increasingly being incorporated into drug discovery screening pipelines for protein misfolding disorders. Together, these diverse screening strategies are generating novel and exciting new probes for understanding the disease process and for furthering development of therapeutic candidates for eventual testing in the clinical setting.     

Methylene Blue Modulates Huntingtin Aggregation Intermediates and Is Protective in Huntington's Disease Models

Huntington's disease (HD) is a devastating neurodegenerative disorder with no disease-modifying treatments available. The disease is caused by expansion of a CAG trinucleotide repeat and manifests with progressive motor abnormalities, psychiatric symptoms, and cognitive decline. Expression of an expanded polyglutamine repeat within the Huntingtin (Htt) protein impacts numerous cellular processes, including protein folding and clearance. A hallmark of the disease is the progressive formation of inclusions that represent the culmination of a complex aggregation process. Methylene blue (MB), has been shown to modulate aggregation of amyloidogenic disease proteins. We investigated whether MB could impact mutant Htt-mediated aggregation and neurotoxicity. MB inhibited recombinant protein aggregation in vitro, even when added to preformed oligomers and fibrils. MB also decreased oligomer number and size and decreased accumulation of insoluble mutant Htt in cells. In functional assays, MB increased survival of primary cortical neurons transduced with mutant Htt, reduced neurodegeneration and aggregation in a Drosophila melanogaster model of HD, and reduced disease phenotypes in R6/2 HD modeled mice. Furthermore, MB treatment also promoted an increase in levels of BDNF RNA and protein in vivo. Thus, MB, which is well tolerated and used in humans, has therapeutic potential for HD.     

Stem cell‐based cell therapy for Huntington disease: A review

Huntington disease (HD) is a devastating neurodegenerative disorder and no proven medical therapy is currently available to mitigate its clinical manifestations. Although fetal neural transplantation has been tried in both preclinical and clinical investigations, the efficacy is not satisfactory. With the recent explosive progress of stem cell biology, application of stem cell‐based therapy in HD is an exciting prospect. Three kinds of stem cells, embryonic stem cells, bone marrow mesenchymal stem cells and neural stem cells, have previously been utilized in cell therapy in animal models of neurological disorders. However, neural stem cells were preferably used by investigators in experimental HD studies, since they have a clear capacity to become neurons or glial cells after intracerebral or intravenous transplantation, and they induce functional recovery. In this review, we summarize the current state of cell therapy utilizing stem cells in experimental HD animal models, and discuss the future considerations for developing new therapeutic strategies using neural stem cells.     

含NURR1基因腺病毒对神经干细胞分化的影响

目的含NURR1基因的重组腺病毒对神经干细胞分化的影响。方法免疫组化观察转染腺病毒后C17·2神经干细胞后分化效率。结果含NURR1基因的重组腺病毒可使C17·2神经干细胞分化后近76%细胞表现为神经元显型。结论含NURR1基因的腺病毒能明显的提高神经干细胞的分化效率。     

Systemic administration of Congo red does not improve motor or cognitive function in R6/2 mice

Huntington's disease (HD) is a progressive neurodegenerative disorder for which there is no treatment. Prior to the onset of symptoms, abnormal protein aggregates (inclusions) are found in neurons in humans and R6/2 mice. It has been suggested that the progression of HD can be slowed or prevented by disruption of the aggregation process. In agreement with this, it has been reported that systemic treatment of R6/2 mice with Congo red caused a reduction in numbers of striatal inclusions and an improvement in motor symptoms and survival [Sanchez, I., Mahlke, C., Yuan, J., 2003. Pivotal role of oligomerization in expanded polyglutamine neurodegenerative disorders. Nature 421, 373-379]. Here we attempted to replicate this study. We extended the experiment to include measurement of the effects of Congo red on cognitive function in R6/2 mice. Congo red treatment failed to ameliorate either motor or cognitive deficits in R6/2 mice. We suggest that this is due to the inability of Congo red to cross the blood-brain barrier. Since it does not improve the behavioural deterioration that is a key feature of HD, Congo red is unlikely to be useful as a therapy for HD.     

Minocycline and doxycycline are not beneficial in a model of Huntington's disease

Huntington's Disease (HD) is an inherited neurological disorder causing movement impairment, personality changes, dementia, and premature death, for which there is currently no effective therapy. The modified tetracycline antibiotic, minocycline, has been reported to ameliorate the disease phenotype in the R6/2 mouse model of HD. Because the tetracyclines have also been reported to inhibit aggregation in other amyloid disorders, we have investigated their ability to inhibit huntingtin aggregation and further explored their efficacy in preclinical mouse trials. We show that tetracyclines are potent inhibitors of huntingtin aggregation in a hippocampal slice culture model of HD at an effective concentration of 30 microM. However, despite achieving tissue levels approaching this concentration by oral treatment of R6/2 mice with minocycline, we observed no clear difference in their behavioral abnormalities, or in aggregate load postmortem. In the light of these new data, we would advise that caution be exercised in proceeding into human clinical trials of minocycline.     

Evaluation of the benzothiazole aggregation inhibitors riluzole and PGL-135 as therapeutics for Huntington's disease

Huntington's disease (HD) is an inherited progressive neurological disorder for which there is no effective therapy. It is caused by a CAG/polyglutamine repeat expansion that leads to abnormal protein aggregation and deposition in the brain. Several compounds have been shown to disrupt the aggregation process in vitro, including a number of benzothiazoles. To further explore the therapeutic potential of the benzothiazole aggregation inhibitors, we assessed PGL-135 and riluzole in hippocampal slice cultures derived from the R6/2 mouse, confirming their ability to inhibit aggregation with an EC50 of 40 microM in this system. Preliminary pharmacological work showed that PGL-135 was metabolically unstable, and therefore, we conducted a preclinical trial in the R6/2 mouse with riluzole. At the maximum tolerated dose, we achieved steady-state riluzole levels of 100 microM in brain. However, this was insufficient to inhibit aggregation in vivo and we found no improvement in the disease phenotype.     

Expression of the Huntington’s disease transgene in neural stem cell cultures from R6/2 transgenic mice

Huntington’s disease (HD) is an inherited neurodegenerative disorder resulting in neuronal cell death in discrete brain regions due to an expanded CAG repeat of the huntingtin gene. The transgenic mouse model R6/2 expresses exon 1 of the human huntingtin gene with >150 CAG repeats, which produces mutant HD protein with an expanded poly-glutamine tract. We have established a neuronal stem cell system deriving from transgenic HD R6/2 neonatal brains as a renewable source for neurons and glia to facilitate studies of HD neuropathology and therapies. These R6/2 stem cell cultures can be cryopreserved and revived. Thawed neural progenitors can be expanded, established as continuous cell lines, and induced to differentiate into glia and neurons. Using standard culture conditions, there was no detectable morphological difference between wild type and HDR6/2 cells. Western analysis reveals that R6/2, but not wild type neurospheres, express the expanded repeat transgenic protein. Immunocytochemistry reveals that at a higher antibody concentration, huntingtin can be localized in the nucleus and the cytoplasm of wild type and R6/2 cells. We conclude that the R6/2 neuronal stem cell culture is a valuable tool for investigating HD pathogenesis and potential genetic or pharmacological interventions.     

Expression of the Huntington's disease transgene in neural stem cell cultures from R6/2 transgenic mice.

Huntington's disease (HD) is an inherited neurodegenerative disorder resulting in neuronal cell death in discrete brain regions due to an expanded CAG repeat of the huntingtin gene. The transgenic mouse model R6/2 expresses exon 1 of the human huntingtin gene with >150 CAG repeats, which produces mutant HD protein with an expanded poly-glutamine tract. We have established a neuronal stem cell system deriving from transgenic HD R6/2 neonatal brains as a renewable source for neurons and glia to facilitate studies of HD neuropathology and therapies. These R6/2 stem cell cultures can be cryopreserved and revived. Thawed neural progenitors can be expanded, established as continuous cell lines, and induced to differentiate into glia and neurons. Using standard culture conditions, there was no detectable morphological difference between wild type and HDR6/2 cells. Western analysis reveals that R6/2, but not wild type neurospheres, express the expanded repeat transgenic protein. Immunocytochemistry reveals that at a higher antibody concentration, huntingtin can be localized in the nucleus and the cytoplasm of wild type and R6/2 cells. We conclude that the R6/2 neuronal stem cell culture is a valuable tool for investigating HD pathogenesis and potential genetic or pharmacological interventions.     

IGF-1 protects against diabetic features in an in vivo model of Huntington's disease

Huntington's disease (HD) is the most prevalent polyglutamine expansion disorder. HD is caused by an expansion of CAG triplet in the huntingtin (HTT) gene, associated with striatal and cortical neuronal loss. Central and peripheral metabolic abnormalities and altered insulin-like growth factor-1 (IGF-1) levels have been described in HD. Thus, we hypothesized that restoration of IGF-1-mediated signaling pathways could rescue R6/2 mice from metabolic stress and behavioral changes induced by polyglutamine expansion. We analyzed the in vivo effect of continuous peripheral IGF-1 administration on diabetic parameters, body weight and motor behavior in the hemizygous R6/2 mouse model of HD. We used 9 week-old and age-matched wild-type mice, subjected to continuously infused recombinant IGF-I or vehicle, for 14 days. IGF-1 treatment prevented the age-related decrease in body weight in R6/2 mice. Although blood glucose levels were higher in R6/2 mice, they did not reach a diabetic state. Even though, IGF-1 ameliorated poor glycemic control in HD mice. This seemed to be associated with a decrease in blood insulin levels in R6/2 mice, which was increased following IGF-1 infusion. Similarly, blood IGF-1 levels decreased during aging in both wild-type and R6/2 mice, being significantly improved upon its continuous infusion. Although no significant differences were found in motor function in R6/2-treated mice, IGF-1 treatment highly improved paw clasping scores. In summary, these results suggest that IGF-1 has a protective role against HD-associated impaired glucose tolerance, by enhancing blood insulin levels.     

The potential of lactulose and melibiose,two novel trehalase-indigestible and autophagy-inducing disaccharides,for polyQ-mediated neurodegenerative disease treatment

The unique property of trehalose encourages its pharmaceutical application in aggregation-mediated neurodegenerative disorders, including Alzheimer's, Parkinson's, and many polyglutamine (polyQ)-mediated diseases. However, trehalose is digested into glucose by trehalase and which reduced its efficacy in the disease target tissues. Therefore, searching trehalase-indigestible analogs of trehalose is a potential strategy to enhance therapeutic effect. In this study, two trehalase-indigestible trehalose analogs, lactulose and melibiose, were selected through compound topology and functional group analyses. Hydrogen-bonding network analyses suggest that the elimination of the hydrogen bond between the linker ether and aspartate 321 (D321) of human trehalase is the key for lactulose and melibiose to avoid the hydrolyzation. Using polyQ-mediated spinocerebellar ataxia type 17 (SCA17) cell and slice cultures, we found the aggregation was significantly prohibited by trehalose, lactulose, and melibiose, which may through up-regulating of autophagy. These findings suggest the therapeutic applications of trehalase-indigestible trehalose analogs in aggregation-associated neurodegenerative diseases.     

Trehalose differentially inhibits aggregation and neurotoxicity of beta-amyloid 40 and 42

A key event in Alzheimer's disease (AD) pathogenesis is the conversion of the peptide beta-amyloid (Abeta) from its soluble monomeric form into various aggregated morphologies in the brain. Preventing aggregation of Abeta is being actively pursued as a primary therapeutic strategy for treating AD. Trehalose, a simple disaccharide, has been shown to be effective in preventing the deactivation of numerous proteins and in protecting cells against stress. Here, we show that trehalose is also effective in inhibiting aggregation of Abeta and reducing its cytotoxicity, although it shows differential effects toward Abeta40 and Abeta42. When co-incubated with Abeta40, trehalose inhibits formation of both fibrillar and oligomeric morphologies as determined by fluorescence staining and atomic force microscopy (AFM). However, when co-incubated with Abeta42, trehalose inhibits formation only of the fibrillar morphology, with significant oligomeric formation still present. When aggregated mixtures were incubated with SH-SY5Y cells, trehalose was shown to reduce the toxicity of Abeta40 mixtures, but not Abeta42. These results provide additional evidence that aggregation of Abeta into soluble oligomeric forms is a pathological step in AD and that Abeta42 in particular is more susceptible to forming these toxic oligomers than Abeta40. These results also suggest that the use of trehalose, a highly soluble, low-priced sugar, as part of a potential therapeutic cocktail to control Abeta peptide aggregation and toxicity warrants further study.     

Neurturin基因的克隆及其在C17.2神经干细胞中表达的研究

目的 探讨Neurturin(NTN)基因转染的C17．2神经干细胞移植后对帕金森病(PD)大鼠模型的保护作用，为其提供基因修饰的神经干细胞。方法 用RT—PCR方法获取Prepro-NTN cDNA，并克隆至pBlueskipt Ⅱ载体中，再将Prepro-NTN基因克隆至pcDNA3．1—hygro真核表达载体中，经量Lipofectamine 2000转染C17．2神经干细胞，挑选稳定表达的克隆，用RT—PCR及Western Blot印迹分析鉴定。结果 pcDNA3．1—hygro-NTN质粒转染C17.2神经干细胞后有5个稳定表达的克隆生长，克隆1有NTN蛋白的高表达。结论 获得了稳定表达NTN蛋白的C17．2神经干细胞克隆，为移植治疗PD打下了坚实的实验基础。     

N-Acetylcysteine improves mitochondrial function and ameliorates behavioral deficits in the R6/1 mouse model of Huntington's disease

Huntington''s disease (HD) is a neurodegenerative disorder, involving psychiatric, cognitive and motor symptoms, caused by a CAG-repeat expansion encoding an extended polyglutamine tract in the huntingtin protein. Oxidative stress and excitotoxicity have previously been implicated in the pathogenesis of HD. We hypothesized that N-acetylcysteine (NAC) may reduce both excitotoxicity and oxidative stress through its actions on glutamate reuptake and antioxidant capacity. The R6/1 transgenic mouse model of HD was used to investigate the effects of NAC on HD pathology. It was found that chronic NAC administration delayed the onset and progression of motor deficits in R6/1 mice, while having an antidepressant-like effect on both R6/1 and wild-type mice. A deficit in the astrocytic glutamate transporter protein, GLT-1, was found in R6/1 mice. However, this deficit was not ameliorated by NAC, implying that the therapeutic effect of NAC is not due to rescue of the GLT-1 deficit and associated glutamate-induced excitotoxicity. Assessment of mitochondrial function in the striatum and cortex revealed that R6/1 mice show reduced mitochondrial respiratory capacity specific to the striatum. This deficit was rescued by chronic treatment with NAC. There was a selective increase in markers of oxidative damage in mitochondria, which was rescued by NAC. In conclusion, NAC is able to delay the onset of motor deficits in the R6/1 model of Huntington''s disease and it may do so by ameliorating mitochondrial dysfunction. Thus, NAC shows promise as a potential therapeutic agent in HD. Furthermore, our data suggest that NAC may also have broader antidepressant efficacy.     

Multimodal treatment for spinal cord injury: a sword of neuroregeneration upon neuromodulation

Spinal cord injury is linked to the interruption of neural pathways,which results in irreversible neural dysfunction.Neural repair and neuroregeneration are critical goals and issues for rehabilitation in spinal cord injury,which require neural stem cell repair and multimodal neuromodulation techniques involving personalized rehabilitation strategies.Besides the involvement of endogenous stem cells in neurogenesis and neural repair,exogenous neural stem cell transplantation is an emerging effective method for repairing and replacing damaged tissues in central nervous system diseases.However,to ensure that endogenous or exogenous neural stem cells truly participate in neural repair following spinal cord injury,appropriate interventional measures(e.g.,neuromodulation)should be adopted.Neuromodulation techniques,such as noninvasive magnetic stimulation and electrical stimulation,have been safely applied in many neuropsychiatric diseases.There is increasing evidence to suggest that neuromagnetic/electrical modulation promotes neuroregeneration and neural repair by affecting signaling in the nervous system;namely,by exciting,inhibiting,or regulating neuronal and neural network activities to improve motor function and motor learning following spinal cord injury.Several studies have indicated that fine motor skill rehabilitation training makes use of residual nerve fibers for collateral growth,encourages the formation of new synaptic connections to promote neural plasticity,and improves motor function recovery in patients with spinal cord injury.With the development of biomaterial technology and biomechanical engineering,several emerging treatments have been developed,such as robots,brain-computer interfaces,and nanomaterials.These treatments have the potential to help millions of patients suffering from motor dysfunction caused by spinal cord injury.However,large-scale clinical trials need to be conducted to validate their efficacy.This review evaluated the efficacy of neural stem cells and magnetic or electrical stimulation combined with rehabilitation training and intelligent therapies for spinal cord injury according to existing evidence,to build up a multimodal treatment strategy of spinal cord injury to enhance nerve repair and regeneration.     

Impaired glutamate uptake in the R6 Huntington's disease transgenic mice

Huntington's disease (HD) is a late-onset neurodegenerative disease for which the mutation is CAG/polyglutamine repeat expansion. The R6 mouse lines expressing the HD mutation develop a movement disorder that is preceded by the formation of neuronal polyglutamine aggregates. The phenotype is likely caused by a widespread neuronal dysfunction, whereas neuronal cell death occurs late and is very selective. We show that a decreased mRNA level of the major astroglial glutamate transporter (GLT1) in the striatum and cortex of these mice is accompanied by a concomitant decrease in glutamate uptake. In contrast, the expression of the glutamate transporters, GLAST and EAAC1, remain unchanged. The mRNA level of the astroglial enzyme glutamine synthetase is also decreased. These changes in expression occur prior to any evidence of neurodegeneration and suggest that a defect in astrocytic glutamate uptake may contribute to the phenotype and neuronal cell death in HD.