Tp53 gene mediates distinct dopaminergic neuronal damage in different dopaminergic neurotoxicant models

Tp53, a stress response gene, is involved in diverse cell death pathways and its activation is implicated in the pathogenesis of Parkinson's disease. However, whether the neuronal Tp53 protein plays a direct role in regulating dopaminergic (DA) neuronal cell death or neuronal terminal damage in different neurotoxicant models is unknown. In our recent studies, in contrast to the global inhibition of Tp53 function by phar-macological inhibitors and in traditional Tp53 knock-out mice, we examined the effects of DA-specific Tp53 gene deletion after 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and methamphetamine exposure. Our data suggests that the Tp53 gene might be involved in both neuronal apoptosis and neuronal termi-nal damage caused by different neurotoxicants. Additional results from other studies also suggest that as a master regulator of many pathways that regulate apoptosis and synaptic terminal damage, it is possible that Tp53 may function as a signaling hub to integrate different signaling pathways to mediate distinctive target pathways. Tp53 protein as a signaling hub might be able to evaluate the microenvironment of neurons, assess the forms and severities of injury incurred, and determine whether apoptotic cell death or neuro-nal terminal degeneration occurs. Identification of the precise mechanisms activated in distinct neuronal damage caused by different forms and severities of injuries might allow for development of specific Tp53 inhibitors or ways to modulate distinct downstream target pathways involved.     

Emerging concepts underlying selective neuromuscular dysfunction in infantile-onset spinal muscular atrophy

Infantile-onset spinal muscular atrophy is the quintessential example of a disorder characterized by a predominantly neurodegenerative phenotype that nevertheless stems from perturbations in a housekeeping protein.Resulting from low levels of the Survival of Motor Neuron(SMN)protein,spinal muscular atrophy manifests mainly as a lower motor neuron disease.Why this is so and whether other cell types contribute to the classic spinal muscular atrophy phenotype continue to be the subject of intense investigation and are only now gaining appreciation.Yet,what is emerging is sometimes as puzzling as it is instructive,arguing for a careful re-examination of recent study outcomes,raising questions about established dogma in the field and making the case for a greater focus on milder spinal muscular atrophy models as tools to identify key mechanisms driving selective neuromuscular dysfunction in the disease.This review examines the evidence for novel molecular and cellular mechanisms that have recently been implicated in spinal muscular atrophy,highlights breakthroughs,points out caveats and poses questions that ought to serve as the basis of new investigations to better understand and treat this and other more common neurodegenerative disorders.     

Autophagy inhibition: a new therapeutic target in spinal muscular atrophy

正Spinal muscular atrophy(SMA)is a hereditary pediatric motor neuron(MN)disease:survival motor neuron 1(SMN1)gene mutation determines MN degeneration and,consequently,muscle atrophy,breathing and swallowing difficulties,and,in the most severe cases,premature death.A second unaffected gene(SMN2)is present,but it can only produce a limited amount of functional protein,modulating the disease severity and progression.SMN,ubiquitously expressed,is mainly involved     

Axonal mRNA localization and local translation in neurodegenerative diseases

The regulation of mRNA localization and local translation play vital roles in the maintenance of cellular structure and function.Many human neurodegenerative diseases,such as fragile X syndrome,amyotrophic lateral sclerosis,Alzheimer’s disease,and spinal muscular atrophy,have been characterized by pathological changes in neuronal axons,including abnormal mRNA translation,the loss of protein expression,or abnormal axon transport.Moreover,the same protein and mRNA molecules have been associated with variable functions in different diseases due to differences in their interaction networks.In this review,we briefly examine fragile X syndrome,amyotrophic lateral sclerosis,Alzheimer’s disease,and spinal muscular atrophy,with a focus on disease pathogenesis with regard to local mRNA translation and axon transport,suggesting possible treatment directions.     

The importance of fasciculation and elongation protein zeta-1 in neural circuit establishment and neurological disorders

The human brain contains an estimated 100 billion neurons that must be systematically organized into functional neural circuits for it to function properly.These circuits range from short-range local signaling networks between neighboring neurons to long-range networks formed between various brain regions.Compelling converging evidence indicates that alterations in neural circuits arising from abnormalities during early neuronal development or neurodegeneration contribute significantly to the etiology of neurological disorders.Supporting this notion,efforts to identify genetic causes of these disorders have uncovered an over-representation of genes encoding proteins involved in the processes of neuronal differentiation,maturation,synaptogenesis and synaptic function.Fasciculation and elongation protein zeta-1,a Kinesin-1 adapter,has emerged as a key central player involved in many of these processes.Fasciculation and elongation protein zeta-1-dependent transport of synaptic cargoes and mitochondria is essential for neuronal development and synapse establishment.Furthermore,it acts downstream of guidance cue pathways to regulate axo-dendritic development.Significantly,perturbing its function causes abnormalities in neuronal development and synapse formation both in the brain as well as the peripheral nervous system.Mutations and deletions of the fasciculation and elongation protein zeta-1 gene are linked to neurodevelopmental disorders.Moreover,altered phosphorylation of the protein contributes to neurodegenerative disorders.Together,these findings strongly implicate the importance of fasciculation and elongation protein zeta-1 in the establishment of neuronal circuits and its maintenance.     

Senegenin inhibits neuronal apoptosis after spinal cord contusion injury

Senegenin has been shown to inhibit neuronal apoptosis, thereby exerting a neuroprotective effect. In the present study, we established a rat model of spinal cord contusion injury using the modiifed Allen’s method. Three hours after injury, senegenin (30 mg/g) was injected into the tail vein for 3 consecutive days. Senegenin reduced the size of syringomyelic cavities, and it substantially reduced the number of apop-totic cells in the spinal cord. At the site of injury, Bax and Caspase-3 mRNA and protein levels were decreased by senegenin, while Bcl-2 mRNA and protein levels were increased. Nerve ifber density was increased in the spinal cord proximal to the brain, and hindlimb motor function and electrophysiological properties of rat hindlimb were improved. Taken together, our results suggest that senegenin exerts a neuroprotective effect by suppressing neuronal apoptosis at the site of spinal cord injury.     

Recent advances in RNA-targeting therapy for neurological diseases

<正>Advances in sequencing and molecular technology now allow us to understand the genetic underpinnings of complex diseases such as neurological disorders.Genetic variations(or mutations) in the DNA sequence of single genes have been implicated in neurological diseases such as Huntington’s disease and spinal muscular atrophy.As a result,the development of gene therapies for neurological diseases is now a feasible endeavor.     

Structural and functional damage to the hippocampal neurovascular unit in diabetes-related depression

Previous studies have shown that models of depression exhibit structural and functional changes to the neurovascular unit. Thus, we hypothesized that diabetes-related depression might be associated with damage to the hippocampal neurovascular unit. To test this hypothesis, neurons, astrocytes and endothelial cells were isolated from the brain tissues of rat embryos and newborn rats. Hippocampal neurovascular unit co-cultures were produced using the Transwell chamber co-culture system. A model of diabetes-related depression was generated by adding 150 mM glucose and 200 μM corticosterone to the culture system and compared with the neuron + astrocyte and astrocyte + endothelial cell co-culture systems. Western blot assay was used to measure levels of structural proteins in the hippocampal neurovascular unit co-culture system. Levels of basic fibroblast growth factor, angiogenic factor 1, glial cell line–derived neurotrophic factor, transforming growth factor β1, leukemia inhibitory factor and 5-hydroxytryptamine in the hippocampal neurovascular unit co-culture system were measured by enzyme-linked immunosorbent assay. Flow cytometry and terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick end labeling staining was used to assess neuronal apoptosis in the hippocampal neurovascular unit. The neurovascular unit triple cell co-culture system had better barrier function and higher levels of structural and secretory proteins than the double cell co-culture systems. In comparison, in the model of diabetes-related depression, the neurovascular unit was damaged with decreased barrier function, poor structural integrity and impaired secretory function. Moreover, neuronal apoptosis was markedly increased, and 5-hydroxytryptamine levels were reduced. These results suggest that diabetes-related depression is associated with structural and functional damage to the neurovascular unit. Our findings provide a foundation for further studies on the pathogenesis of diabetes-related depression.     

Scutellaria baicalensis stem-leaf total flavonoid reduces neuronal apoptosis induced by amyloid beta-peptide (25-35)

Scutellaria baicalensis stem-leaf total flavonoid might attenuate learning/memory impairment and neuronal loss in rats induced by amyloid beta-peptide. This study aimed to explore the effects of Scutellaria baicalensis stem-leaf total flavonoid on amyloid beta-peptide-induced neuronal apoptosis and the expression of apoptosis-related proteins in the rat hippocampus. Male Wistar rats were given intragastric administration of Scutellaria baicalensis stem-leaf total flavonoid, 50 or 100 mg/kg, once per day. On day 8 after administration, 10 μg amyloid beta-peptide (25-35) was injected into the bilateral hippocampus of rats to induce neuronal apoptosis. On day 20, hippocampal tissue was harvested and probed with the terminal deoxyribonucleotidyl transferase-mediated biotin-16-dUTP nick-end labeling assay. Scutellaria baicalensis stem-leaf total flavonoid at 50 and 100 mg/kg reduced neuronal apoptosis induced by amyloid beta-peptide (25-35 in the rat hippocampus. Immunohistochemistry and western blot assay revealed that expression of the pro-apoptotic protein Bax, cytochrome c and caspase-3 was significantly diminished by 50 and 100 mg/kg Scutellaria baicalensis stem-leaf total flavonoid, while expression of the anti-apoptotic protein Bcl-2 was increased. Moreover, 100 mg/kg Scutellaria baicalensis stem-leaf total flavonoid had a more dramatic effect than the lower dosage. These experimental findings indicate that Scutellaria baicalensis stem-leaf total flavonoid dose-dependently attenuates neuronal apoptosis induced by amyloid beta-peptide in the hippocampus, and it might mediate this by regulating the expression of Bax, cytochrome c, caspase-3 and Bcl-2.     

ClC-3 chloride channel in hippocampal neuronal apoptosis

Over-production of nitric oxide is pathogenic for neuronal apoptosis around the ischemic area fol- lowing ischemic brain injury. In this study, an apoptotic model in rat hippocampal neurons was es- tablished by 0.5 mmol/L 3-morpholinosyndnomine （SIN-l）, a nitric oxide donor. The models were then cultured with 0.1 mmol/L of 4,4＇-diisothiocyanostilbene-2,2＇-disulfonic acid （DIDS; the chloride channel blocker）for 18 hours. Neuronal survival was detected using the 3-（4,5-dimethylthiazol- 2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay, and apoptosis was assayed by Hoechst 33342-labeled neuronal DNA fluorescence staining. Western blot analysis and immunochemilumi- nescence staining were applied to determine the changes of activated caspase-3 and CIC-3 channel proteins. Real-time PCR was used to detect the mRNA expression of CIC-3. The results showed that SIN-1 reduced the neuronal survival rate, induced neuronal apoptosis, and promoted CIC-3 chloride channel protein and mRNA expression in the apoptotic neurons. DIDS reversed the effect of SIN-I. Our findings indicate that the increased activities of the CIC-3 chloride channel may be involved in hippocampal neuronal apoptosis induced by nitric oxide.     

High incidence of SMN1 gene deletion in Moroccan adult-onset spinal muscular atrophy patients

Abstract. Spinal muscular atrophy (SMA) is an autosomal recessive motor neuropathy characterized by selective degeneration of anterior horn cells of the spinal cord. Childhood SMA is divided into three types (I–III) on the basis of age of onset and severity. These disorders have been linked to the 5q13 region, where mutations in the Survival Motor Neuron 1 (SMN1) gene have been found in affected individuals. In the case of adult-onset SMA (type IV), on the other hand, reports of homozygous absence of SMN1 gene have been rare. We conducted deletion analysis of SMN and a neighboring gene, NAIP (neuronal apoptosis inhibiting protein). Among 54SMA patients (types I–IV), all of Moroccan origin, Exon 7 of the SMN1 gene was homozygously absent in 100% of type I, 90% of type II, 74% of type III and 80% of type IV SMA patients. Deletion of SMN1 exon 8 was detected in 100% of type I, 53% of type II, 53% of type III and 80% of type IV patients. NAIP exon 5 was homozygously deleted in 67% of type I, 32% of type II, 5% of type III and 20% of type IV SMA patients. Thirty control individuals who were studied had normal SMN1 and NAIP genes. Our results show a high incidence of SMN1 gene deletion in adult-onset SMA patients indicating that SMN1 is the autosomal recessive adult SMA-causing gene. While NAIP is commonly deleted in SMA, this is unlikely to affect disease severity; it was deleted in two adult SMA patients with mild phenotypes.     

Novel point mutations in survival motor neuron 1 gene expand the spectrum of phenotypes observed in spinal muscular atrophy patients

The aim of our study was to identify point mutations in a group of 606 patients diagnosed for spinal muscular atrophy with excluded biallelic loss of the SMN1 gene. Point missense mutations or small deletions in the SMN1 gene were ultimately identified in 18 patients. Six patients were found to have small deletions, the c.429_435del mutation in 3 cases, the c.431delC mutation in 2 and c.722delC in one. Those mutations, not described previously, were characteristic of patients presenting a severe phenotype. The most frequent missense mutation – p.Thr274Ile, was identified in 9 patients presenting a rather mild phenotype. Three other missense mutations, i.e. p.Ser230Leu, p.Ala111Gly and p.Pro244Leu, were identified in a further 3 SMA3 patients. Mutation p.Pro244Leu, not described so far, was identified in a patient with a mild form of SMA and more distal distribution of muscle weakness. Our results suggest a specific point mutation spectrum in the Polish population. The existence of small deletions not identified thus far could suggest a possible founder effect. In patients with preserved one SMN1 allele without common exon 7 deletion, presenting a mild form of SMA, a special consideration should be given to the p.Thr274Ile mutation.     

应用多重连接依赖性探针扩增技术进行脊髓性肌萎缩症产前诊断

目的 探讨多重连接依赖性探针扩增技术在脊髓性肌萎缩症产前诊断中的临床应用价值.方法 以脊髓性肌萎缩症6个家系作为研究对象.包括患者7例、父母12名、胎儿6例.采用多重连接依赖性探针扩增技术对运动神经元生存(SMN)基囚及脊髓性肌萎缩症修饰基因进行分析,应用聚合酶链反应-限制性酶切片段长度多态性技术检测SMNI基凶缺失,羊水标本分别通过直接离心沉淀和细胞培养进行DNA分析.结果 多重连接依赖性探针扩增分析提示6个家系中7例患者及1例胎儿(家系Ⅳ)呈SMNI基凶纯合缺失,与聚合酶链反应.限制性酶切片段长度多态性分析结果一致;11名父母及5例胎儿的SMNI拷贝数为1,1名母亲(家系V)SMNI拷贝数为2,均为脊髓性肌萎缩症携带者.多重连接依赖性探针扩增分析显示.6个家系中10名成员SMN2拷贝数为1,15名成员SMN2拷贝数为2;多重连接依赖性探针扩增分析.6个家系中3名成员神经元凋亡抑制蛋白(NAIP)基冈缺失,其余家系成员正常.结论 多重连接依赖性探针扩增技术为一快速而可靠的基凶榆测及定量分析方法,可准确检测SMN基凶及脊髓性肌萎缩症修饰基凶的缺失突变并分析基因拷贝数,适用于脊髓性肌萎缩症患者、携带者的基因诊断及产前诊断.     

Gene conversion events in adult-onset spinal muscular atrophy

OBJECTIVE: To investigate the possible occurrence of a conversion event in three patients with adult-onset spinal muscular atrophy (SMA) type IV, which represents the mildest form within the spectrum of the SMA phenotype. MATERIAL AND METHODS: We observed three patients with adult onset SMA and apparent isolated deletion of telomeric survival motor neuron (SMN1) exon 7. To distinguish between a deletion and a sequence conversion event of exon 7, these patients were analyzed in greater detail by a simple PCR-based assay. RESULTS: Analysis by DdeI digestion showed products for both telomeric and centromeric copies of exon 8. These findings indicated a gene conversion event as the site for primer R111 was retained at least in one of two alleles. CONCLUSIONS: These results provide first evidence that a conversion event may be also associated with adult-onset SMA, and further support the notion that a gene conversion event is usually associated with a milder SMA phenotype and a later onset of disease.     

Deletion analysis in Turkish patients with spinal muscular atrophy

Childhood proximal spinal muscular atrophy (SMA) is an autosomal recessive disorder which presents as a severe, intermediate or mild condition. Here we present the molecular analysis of SMA candidate genes, the survival motor neuron gene (SMN), the neuronal apoptosis inhibitory protein gene (NAIP) and the p44 gene. Deletion frequency rate of these candidate genes is 93% in 106 Turkish SMA patients. Various deletion haplotypes by using genotypes of SMN, NAIP and p44 genes are constructed. Haplotype A, which is the deletion of all three involved genes, was found only in the most severe group with an early onset of usually less than 2 months of age.     

Utility and intricacy of molecular diagnosis of spinal muscular atrophy

To diagnose spinal muscular atrophy (SMA), we examined the deletion of exons 7 and 8 of the survival motor neuron (SMN) gene and exon 5 of the neuronal apoptosis inhibitory protein (NAIP) gene in 7 patients from 6 unrelated families, using the polymerase chain reaction method. Two patients with type I and two with type II SMA had the deletion in SMN, whereas 2 of the 3 patients with type III had no deletion in these genes. Thus, the method was not as useful in type III as in type I and II for making a diagnosis of SMA. Together with the data previously reported by others, our data indicated the possibility that the deletion frequency in type III SMA is lower in Japanese patients (< 40%) than in non-Japanese patients (> 80%). Two siblings had SMA of different severity; the older brother having type III and the younger brother type II. Both had the same deletion in the SMN gene. The different phenotypes in these siblings with the same genotype indicated that caution is required when utilizing molecular data for genetic counseling or prenatal diagnosis of SMA.     

Clinical phenotypes of spinal muscular atrophy patients with hybrid SMN gene

BackgroundSpinal muscular atrophy (SMA) is a neuromuscular disease caused by homozygous deletion of SMN1 exons 7 and 8. However, exon 8 is retained in some cases, where SMN2 exon 7 recombines with SMN1 exon 8, forming a hybrid SMN gene. It remains unknown how the hybrid SMN gene contribute to the SMA phenotype.MethodWe analyzed 515 patients with clinical suspicion for SMA. SMN1 exons 7 and 8 deletion was detected by PCR followed by enzyme digestion. Hybrid SMN genes were further analyzed by nucleotide sequencing. SMN2 copy number was determined by real-time PCR.ResultsSMN1 exon 7 was deleted in 228 out of 515 patients, and SMN1 exon 8 was also deleted in 204 out of the 228 patients. The remaining 24 patients were judged to carry a hybrid SMN gene. In the patients with SMN1 exon 7 deletion, the frequency of the severe phenotype was significantly lower in the patients with hybrid SMN gene than in the patients without hybrid SMN gene. However, as for the distribution of SMN2 exon 7 copy number among the clinical phenotypes, there was no significant difference between both groups of SMA patients with or without hybrid SMN gene.ConclusionHybrid SMN genes are not rare in Japanese SMA patients, and it appears to be associated with a less severe phenotype. The phenotype of patients with hybrid SMN gene was determined by the copy number of SMN2 exon 7, as similarly for the patients without hybrid SMN gene.     

SMN蛋白与脊髓性肌萎缩症的研究进展

本文着重就SMN蛋白分布、结构、功能及其与脊髓性肌萎缩症 (SMA)的关系进行了综述。而深入研究SMN蛋白的表达及其功能 ,对于深入研究SMA的发病机制有重要意义。调控SMN2基因表达 ,促进全长SMN蛋白产生 ,有望为治疗SMA开辟新途径。     

Plastin 3 expression in discordant spinal muscular atrophy (SMA) siblings

Spinal muscular atrophy (SMA) is caused by loss or mutations of the survival motor neuron 1 gene (SMN1). Its highly homologous copy, SMN2, is present in all SMA cases and is a phenotypic modifier. There are cases where asymptomatic siblings of typical SMA patients possess a homozygous deletion of SMN1 just like their symptomatic brothers or sisters. Plastin 3 (PLS3) when over expressed in lymphoblasts from females has been suggested to act as a genetic modifier of SMA.We studied PLS3 expression in four Spanish SMA families with discordant siblings haploidentical for the SMA locus. We excluded PLS3 as a possible modifier in two of our families with female discordant siblings. In the remaining two, we observed small differences in PLS3 expression between male and female discordant siblings. Indeed, we found that values of PLS3 expression in lymphoblasts and peripheral blood ranged from 12 to 200-fold less than those in fibroblasts. These findings warrant further investigation in motor neurons derived from induced pluripotential stem cells of these patients.     

Pathogenesis of proximal autosomal recessive spinal muscular atrophy

Although it is known that deletions or mutations of the SMN1 gene on chromosome 5 cause decreased levels of the SMN protein in subjects with proximal autosomal recessive spinal muscular atrophy (SMA), the exact sequence of pathological events leading to selective motoneuron cell death is not fully understood yet. In this review, new findings regarding the dual cellular role of the SMN protein (translocation of beta-actin to axonal growth cones and snRNP biogenesis/pre-mRNA splicing) were integrated with recent data obtained by detailed neuropathological examination of SMA and control subjects. A presumptive series of 10 pathogenetic events for SMA is proposed as follows: (1) deletions or mutations of the SMN1 gene, (2) increased SMN mRNA decay and reduction in full-length functional SMN protein, (3) impaired motoneuron axono- and dendrogenesis, (4) failure of motoneurons to form synapses with corticospinal fibers from upper motoneurons, (5) abnormal motoneuron migration towards ventral spinal roots, (6) inappropriate persistence of motoneuron apoptosis due to impaired differentiation and motoneuron displacement, (7) substantial numbers of motoneurons continuing to migrate abnormally ("heterotopic motoneurons") and entering into the ventral roots, (8) attracted glial cells following these heterotopic motoneurons, which form the glial bundles of ventral roots, (9) impaired axonal transport of actin, causing remaining motoneurons to become chromatolytic, and (10) eventual death of all apoptotic, heterotopic and chromatolytic neurons, with apoptosis being more rapid and predominating in the earlier stages, with death of heterotopic and chromatolytic neurons occurring more slowly by necrosis during the later stages of SMA. According to this model, the motoneuron axonopathy is more important for pathogenesis than the ubiquitous nuclear splicing deficit. It is also supposed that individually variable levels of SMN protein, together with influences of other phenotype modifier genes and their products, cause the clinical SMA spectrum through differential degree of motoneuron functional loss.