PCR片段测序和基因分型两种方法对Kennedy遗传病的诊断

X连锁脊延髓肌萎缩症（SBMA）或肯尼迪病是一种成年人发病的神经变性疾病,以肌无力与慢性、进行性肌萎缩为特征. 通过PCR片段测序和基因分型法准确检测雄激素受体（AR）基因CAG复制数目,兄弟俩（来自同一个中国家庭）被确诊为隐性遗传性SBMA. 为了得到该中国家庭SMBA家系人员AR基因的CAG复制数目,我们采用了PCR片段测序和基因分型两种方法. 在该SMBA家系中有两个已发病的成年男性、未发病的年轻男性,及女性基因携带者. 两个已发病男性患者AR基因中CAG三核苷酸串重复数目分别是48和45. 以前的研究表明特定三核苷酸串重复数目的扩增可导致人类遗传性神经障碍疾病发病。我们的研究结果完全支持这一观点,SMBA中国家系的三核苷酸CAG拷贝数目检测结果表明,AR基因CAG扩增数目与SMBA发病相关. 关键词雄性激素受体; CAG多重三核苷酸重复; 肯尼迪病; 脊延髓肌萎缩症; X连锁     

Biophysical Underpinnings of the Repeat Length Dependence of Polyglutamine Amyloid Formation

There are now 10 expanded CAG repeat diseases in which both disease risk and age of onset are strongly dependent on the repeat length of the polyglutamine (polyQ) sequence in the disease protein. Large, polyQ-rich inclusions in patient brains and in cell and animal models are consistent with the involvement of polyQ aggregation in the disease mechanism. This possibility is reinforced by studies showing strong repeat length dependence to the aggregation process, qualitatively mirroring the repeat length dependence of disease risk. Our understanding of the underlying biophysical principles that mediate the repeat length dependence of aggregation, however, is far from complete. A previous study of simple polyQ peptides showed that N*, the size of the critical nucleus that controls onset of aggregation, decreases from unfavorable tetramer to favorable monomer over the range Q₂₃ to Q₂₆. These data, however, do not explain why, for all peptides exhibiting N* ∼ 1, spontaneous aggregation rates continue to increase with increasing repeat length. Here we describe a novel kinetics analyses that maps out the nonlinear dependence with repeat length of a nucleation efficiency term that is likely related to aspects of nucleus structure. This trend accounts for why nucleus size increases to tetrameric at repeat lengths of Q₂₃ or below. Intriguingly, both aggregation and age of onset trend with repeat length in similar ways, exhibiting large changes per added Gln at low repeat lengths and small changes per added Gln at relatively long repeat lengths. Fibril stability also increases with repeat length in a nonlinear fashion.     

Pathogenesis of inclusion bodies in (CAG)n/Qn-expansion diseases with special reference to the role of tissue transglutaminase and to selective vulnerability

At least eight neurodegenerative diseases, including Huntington disease, are caused by expansions in (CAG)n repeats in the affected gene and by an increase in the size of the corresponding polyglutamine domain in the expressed protein. A hallmark of several of these diseases is the presence of aberrant, proteinaceous aggregates in the nuclei and cytosol of affected neurons. Recent studies have shown that expanded polyglutamine (Qn) repeats are excellent glutaminyl-donor substrates of tissue transglutaminase, and that the substrate activity increases with increasing size of the polyglutamine domain. Tissue transglutaminase is present in the cytosol and nuclear fractions of brain tissue. Thus, the nuclear and cytosolic inclusions in Huntington disease may contain tissue transglutaminase-catalyzed covalent aggregates. The (CAG)n/Qn-expansion diseases are classic examples of selective vulnerability in the nervous system, in which certain cells/structures are particularly susceptible to toxic insults. Quantitative differences in the distribution of the brain transglutaminase(s) and its substrates, and in the activation mechanism of the brain transglutaminase(s), may explain in part selective vulnerability in a subset of neurons in (CAG)n-expansion diseases, and possibly in other neurodegenerative disease. If tissue transglutaminase is found to be essential for development of pathogenesis, then inhibitors of this enzyme may be of therapeutic benefit.     

Histone H3 is aberrantly phosphorylated in glutamine-repeat diseases

Double-labeling immunohistochemical studies staining with anti-ubiquitin and anti-phosphoserine antibodies and application of an enzymatic dephosphorylation technique reveal neuronal inclusions and affected nuclei to be aberrantly phosphorylated in brain tissues with patients with glutamine-repeat diseases. Regional distribution of the phosphorylated nuclei in neurons correlates with the pathology. To identify the target nuclear protein, transient expression of Huntington's disease exon 1 gene containing an expanded glutamine repeat was generated in a cell culture and nuclear inclusions were isolated with a fluorescence-activated cell sorting system. Immunoblotting studies of the aggregated nuclear proteins using anti-phosphoserine antibody demonstrate the protein of the aberrant phosphorylation as histone H3. The immunoblots of control and diseased brain tissues demonstrate that the phosphorylation of histone H3 is commonly increased in the diseased brains. Aberrant phosphorylation of histone H3 is surmised to be a shared pathological process in glutamine-repeat diseases.     

A Cell Culture Model for Androgen Effects in Motor Neurons

Abstract: Androgens are known to alter the morphology, survival, and axonal regeneration of lower motor neurons in vivo. To understand better the molecular mechanisms of androgen action in neurons, we created a model system by stably expressing the human androgen receptor (AR) in motor neuron hybrid cells. Motor neuron hybrid cells express markers consistent with anterior horn cells and can be differentiated into a neuronal phenotype. When differentiated in the presence of androgen, AR-expressing cells, but not control cells, exhibit a dose-dependent change in morphology: androgen-treated cells develop larger cell bodies and broader neuritic processes while continuing to express neuronal markers. In addition, androgen promotes the survival of AR-expressing cells, but not control cells, under low-serum conditions. Our results demonstrate a direct trophic effect of androgens on lower motor neurons, mediated through the AR expressed in this population of neurons.     

Polyglutamine expansion inhibits respiration by increasing reactive oxygen species in isolated mitochondria

Huntington’s disease results from expansion of the polyglutamine (PolyQ) domain in the huntingtin protein. Although the cellular mechanism by which pathologic-length PolyQ protein causes neurodegeneration is unclear, mitochondria appear central in pathogenesis. We demonstrate in isolated mitochondria that pathologic-length PolyQ protein directly inhibits ADP-dependent (state 3) mitochondrial respiration. Inhibition of mitochondrial respiration by PolyQ protein is not due to reduction in the activities of electron transport chain complexes, mitochondrial ATP synthase, or the adenine nucleotide translocase. We show that pathologic-length PolyQ protein increases the production of reactive oxygen species in isolated mitochondria. Impairment of state 3 mitochondrial respiration by PolyQ protein is reversed by addition of the antioxidants N-acetyl-l-cysteine or cytochrome c. We propose a model in which pathologic-length PolyQ protein directly inhibits mitochondrial function by inducing oxidative stress.     

Glutamate Uptake is Reduced in Prefrontal Cortex in Huntington’s Disease

Huntington’s disease (HD) is caused by a CAG repeat expansion in the HD gene, but how this mutation causes neuronal dysfunction and degeneration is unclear. Inhibition of glutamate uptake, which could cause excessive stimulation of glutamate receptors, has been found in animals carrying very long CAG repeats in the HD gene. In seven HD patients with moderate CAG expansions (40–52), repeat expansion and HD grade at autopsy were strongly correlated (r = 0.88, p = 0.0002). Uptake of [³H]glutamate was reduced by 43% in prefrontal cortex, but the level of synaptic (synaptophysin, AMPA receptors) and astrocytic markers (GFAP, glutamate transporter EAAT1) were unchanged. Glutamate uptake correlated inversely with CAG repeat expansion (r = −0.82, p = 0.015). The reducing agent dithiothreitol improved glutamate uptake in controls, but not in HD brains, suggesting irreversible oxidation of glutamate transporters in HD. We conclude that impairment of glutamate uptake may contribute to neuronal dysfunction and degeneration in HD. Special issue article in honor of Dr. Frode Fonnum.     

Polyglutamine shows a urea-like affinity for unfolded cytosolic protein

Noting that the glutamine (Q) amino acid side-chain bears a striking resemblance to urea, the chemical denaturant, we argue on biophysical grounds that polyQ chains should possess a potent denaturant activity. Using live-cell confocal microscopy, we demonstrate that the surface of a polyQ inclusion denatures cytosolic proteins by binding and trapping them in an immobilized ring. We also show the reverse effect: that elevated local concentrations of unfolded protein in the cytosol can drive the co-localization and accumulation of short polyQ tracts that normally do not aggregate. Such a urea-like mechanism explains many past observations about polyQ-driven disruption of proteostasis and neurodegeneration.     

Expanding therapeutic opportunities for neurodegenerative diseases: A perspective on the important role of phenotypic screening

Over the last 20 years, there have been remarkably few FDA-approved first-in-class drugs for neurodegenerative diseases. Debilitating conditions such as Alzheimer’s disease, Parkinson’s disease, and amyotrophic lateral sclerosis have no effective disease-modifying therapeutics on the market, signifying an area of high unmet medical need where novel approaches are needed. Using a phenotypic screening approach, two separate groups discovered small molecule non-antisense oligonucleotide splice modulators for spinal muscular atrophy, a severe monogenetic disease that causes the degeneration of alpha motor neurons in the spinal cord. These compounds function by a novel mechanism: selective stabilization of the interaction of U1 small nuclear ribonucleic protein (snRNP), a core component of the spliceosome, with the 5′ splice site of a pre-mRNA. The ability of the phenotypic screening approach to uncover a previously unknown mechanism and reveal a new druggable target class has broader implications for other neurodegenerative diseases.     

Local protein unfolding and pathogenesis of polyglutamine-expansion diseases

The CAPRI Challenge is a blind test of protein-protein-docking algorithms that predict the complex structure from the crystal structures of the interacting proteins. We participated in both rounds of this blind test and submitted predictions for all seven targets, relying mainly on our Fast Fourier Transform based algorithm ZDOCK that combines shape complementarity, desolvation, and electrostatics. Our group made good predictions for three targets and had at least some success with three others. Implications of the treatment of prior biological information as well as contributions of manual inspection to docking predictions are also discussed.     

The lysosome and neurodegenerative diseases

It has long been believed that the lysosome is an important digestive organelle. There is increasing evidence that the lysosome is also involved in pathogenesis of a variety of neurodegenerative diseases, including Alzheimer＇s disease, Parkinson＇s disease, Huntington＇s disease, and amyotrophic lateral sclerosis. Abnormal protein degradation and deposition induced by iysosomal dysfunction may be the primary contributor to age-related neurodegeneration. In this review, the possible relationship between lysosome and various neurodegenerative diseases is described.     

The Possible Structural Models for Polyglutamine Aggregation: A Molecular Dynamics Simulations Study

Abstract

Huntington's disease is a neurodegenerative disorder caused by a polyglutamine (polyQ) expansion near the N-terminus of huntingtin. Previous studies have suggested that polyQ aggregation occurs only when the number of glutamine (Q) residues is more than 36-40, the disease threshold. However, the structural characteristics of polyQ nucleation in the very early stage of aggregation still remain elusive. In this study, we designed 18 simulation trials to determine the possible structural models for polyQ nucleation and aggregation with various shapes and sizes of initial β-helical structures, such as left-handed circular, right-handed rectangular, and left- and right-handed triangular. Our results show that the stability of these models significantly increases with increasing the number of rungs, while it is rather insensitive to the number of Qs in each rung. In particular, the 3-rung β-helical models are stable when they adopt the left-handed triangular and right-handed rectangular conformations due to the fact that they preserve high β-turn and β-sheet contents, respectively, during the simulation courses. Thus, we suggested that these two stable β-helical structures with at least 3 rungs might serve as the possible nucleation seeds for polyQ depending on how the structural elements of β-turn and β-sheet are sampled and preserved during the very early stage of aggregation.     

Polyglutamine expansion diseases: More than simple repeats

Polyglutamine (polyQ) repeat-containing proteins are widespread in the human proteome but only nine of them are associated with highly incapacitating neurodegenerative disorders. The genetic expansion of the polyQ tract in disease-related proteins triggers a series of events resulting in neurodegeneration. The polyQ tract plays the leading role in the aggregation mechanism, but other elements modulate the aggregation propensity in the context of the full-length proteins, as implied by variations in the length of the polyQ tract required to trigger the onset of a given polyQ disease. Intrinsic features such as the presence of aggregation-prone regions (APRs) outside the polyQ segments and polyQ-flanking sequences, which synergistically participate in the aggregation process, are emerging for several disease-related proteins. The inherent polymorphic structure of polyQ stretches places the polyQ proteins in a central position in protein–protein interaction networks, where interacting partners may additionally shield APRs or reshape the aggregation course. Expansion of the polyQ tract perturbs the cellular homeostasis and contributes to neuronal failure by modulating protein–protein interactions and enhancing toxic oligomerization. Post-translational modifications further regulate self-assembly either by directly altering the intrinsic aggregation propensity of polyQ proteins, by modulating their interaction with different macromolecules or by modifying their withdrawal by the cell quality control machinery. Here we review the recent data on the multifaceted aggregation pathways of disease-related polyQ proteins, focusing on ataxin-3, the protein mutated in Machado-Joseph disease. Further mechanistic understanding of this network of events is crucial for the development of effective therapies for polyQ diseases.     

The pathogenic mechanisms of polyglutamine diseases and current therapeutic strategies

Expansion of CAG trinucleotide repeat within the coding region of several genes results in the production of proteins with expanded polyglutamine (PolyQ) stretch. The expression of these pathogenic proteins leads to PolyQ diseases, such as Huntington's disease or several types of spinocerebellar ataxias. This family of neurodegenerative disorders is characterized by constant progression of the symptoms and molecularly, by the accumulation of mutant proteins inside neurons causing their dysfunction and eventually death. So far, no effective therapy actually preventing the physical and/or mental decline has been developed. Experimental therapeutic strategies either target the levels or processing of mutant proteins in an attempt to prevent cellular deterioration, or they are aimed at the downstream pathologic effects to reverse or ameliorate the caused damages. Certain pathomechanistic aspects of PolyQ disorders are discussed here. Relevance of disease models and recent knowledge of therapeutic possibilities is reviewed and updated.     

Influence of species differences on the neuropathology of transgenic Huntington's disease animal models

Transgenic animal models have revealed much about the pathogenesis of age-dependent neurodegenerative diseases and proved to be a useful tool for uncovering therapeutic targets.Huntington's disease is ...     

Surface electromyograph activity of submental muscles during swallowing and expiratory muscle training tasks in Huntington’s disease patients

IntroductionHuntington’s disease (HD) patients have difficulty in swallowing, leading to aspiration pneumonia, which is a major cause of death. It seems possible that submental muscles that are crucial for preventing an escape of a bolus into the airway, are affected by HD, but no previous studies have investigated this.ObjectiveTo assess surface electromyograph (sEMG) activity of submental muscles during swallowing and expiratory muscle training (EMT) tasks in HD patients in comparison to healthy volunteers.MethodssEMG activities of submental muscles during saliva, water swallowing, EMT tasks performed at 25% and 75% of maximum expiratory pressure were recorded and normalised by the sEMG activity during an effortful swallow in 17 early to mid stage HD patients and 17 healthy volunteers.ResultssEMG activity was greater (p < 0.05) during EMT tasks than saliva and water swallowing, but was not significantly different between groups for saliva, water swallowing and EMT at 25%. HD patients had lower sEMG activity for EMT at 75% (p < 0.05).ConclusionDecreases in submental muscle activity were not evident in HD patients except during EMT at 75%. This suggests that relative submental muscle weakness is observed only during a high intensity task in early to mid stage HD patients.