Mimicking Aspects of Frontotemporal Lobar Degeneration and Lou Gehrig's Disease in Rats via TDP-43 Overexpression

Since the discovery of neuropathological lesions made of TDP-43 and ubiquitin proteins in cases of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS), there is a burst of effort on finding related familial mutations and developing animal models. We used an adeno-associated virus (AAV) vector for human TDP-43 expression targeted to the substantia nigra (SN) of rats. Though TDP-43 was expressed mainly in neuronal nuclei as expected, it was also expressed in the cytoplasm, and dotted along the plasma membrane of neurons. Cytoplasmic staining was both diffuse and granular, indicative of preinclusion lesions, over 4 weeks. Ubiquitin deposited in the cytoplasm, specifically in the TDP-43 group, and staining for microglia was increased dose-dependently by 1–2 logs in the TDP-43 group, while neurons were selectively obliterated. Neuronal death induced by TDP-43 was pyknotic and apoptotic. TDP-43 gene transfer caused loss of dopaminergic neurons in the SN and their axons in the striatum. Behavioral motor dysfunction resulted after TDP-43 gene transfer that was vector dose-dependent and progressive over time. The cytoplasmic expression, ubiquitination, and neurodegeneration mimicked features of the TDP-43 diseases, and the gliosis, apoptosis, and motor impairment may also be relevant to TDP-43 disease forms involving nigrostriatal degeneration.     

Dysregulation of the ALS-associated gene TDP-43 leads to neuronal death and degeneration in mice

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are characterized by cytoplasmic protein aggregates in the brain and spinal cord that include TAR-DNA binding protein 43 (TDP-43). TDP-43 is normally localized in the nucleus with roles in the regulation of gene expression, and pathological cytoplasmic aggregates are associated with depletion of nuclear protein. Here, we generated transgenic mice expressing human TDP-43 with a defective nuclear localization signal in the forebrain (hTDP-43-ΔNLS), and compared them with mice expressing WT hTDP-43 (hTDP-43-WT) to determine the effects of mislocalized cytoplasmic TDP-43 on neuronal viability. Expression of either hTDP-43-ΔNLS or hTDP-43-WT led to neuron loss in selectively vulnerable forebrain regions, corticospinal tract degeneration, and motor spasticity recapitulating key aspects of FTLD and primary lateral sclerosis. Only rare cytoplasmic phosphorylated and ubiquitinated TDP-43 inclusions were seen in hTDP-43-ΔNLS mice, suggesting that cytoplasmic inclusions were not required to induce neuronal death. Instead, neurodegeneration in hTDP-43 and hTDP-43-ΔNLS-expressing neurons was accompanied by a dramatic downregulation of the endogenous mouse TDP-43. Moreover, mice expressing hTDP-43-ΔNLS exhibited profound changes in gene expression in cortical neurons. Our data suggest that perturbation of endogenous nuclear TDP-43 results in loss of normal TDP-43 function(s) and gene regulatory pathways, culminating in degeneration of selectively vulnerable affected neurons.     

Mutant TDP-43 in motor neurons promotes the onset and progression of ALS in rats

Amyotrophic lateral sclerosis (ALS) is characterized by progressive motor neuron degeneration, which ultimately leads to paralysis and death. Mutation of TAR DNA binding protein 43 (TDP-43) has been linked to the development of an inherited form of ALS. Existing TDP-43 transgenic animals develop a limited loss of motor neurons and therefore do not faithfully reproduce the core phenotype of ALS. Here, we report the creation of multiple lines of transgenic rats in which expression of ALS-associated mutant human TDP-43 is restricted to either motor neurons or other types of neurons and skeletal muscle and can be switched on and off. All of these rats developed progressive paralysis reminiscent of ALS when the transgene was switched on. Rats expressing mutant TDP-43 in motor neurons alone lost more spinal motor neurons than rats expressing the disease gene in varying neurons and muscle cells, although these rats all developed remarkable denervation atrophy of skeletal muscles. Intriguingly, progression of the disease was halted after transgene expression was switched off; in rats with limited loss of motor neurons, we observed a dramatic recovery of motor function, but in rats with profound loss of motor neurons, we only observed a moderate recovery of motor function. Our finding suggests that mutant TDP-43 in motor neurons is sufficient to promote the onset and progression of ALS and that motor neuron degeneration is partially reversible, at least in mutant TDP-43 transgenic rats.     

Expansive Gene Transfer in the Rat CNS Rapidly Produces Amyotrophic Lateral Sclerosis Relevant Sequelae When TDP-43 is Overexpressed

Improved spread of transduction in the central nervous system (CNS) was achieved from intravenous administration of adeno-associated virus serotype-9 (AAV9) to neonatal rats. Spinal lower motor neuron transduction efficiency was estimated to be 78% using the highest vector dose tested at a 12-week interval. The widespread expression could aid studying diseases that affect both the spinal cord and brain, such as amyotrophic lateral sclerosis (ALS). The protein most relevant to neuropathology in ALS is transactive response DNA-binding protein 43 (TDP-43). When expressed in rats, human wild-type TDP-43 rapidly produced symptoms germane to ALS including paralysis of the hindlimbs and muscle wasting, and mortality over 4 weeks that did not occur in controls. The hindlimb atrophy and weakness was evidenced by assessments of rotarod, rearing, overall locomotion, muscle mass, and histology. The muscle wasting suggested denervation, but there was only 14% loss of motor neurons in the TDP-43 rats. Tissues were negative for ubiquitinated, cytoplasmic TDP-43 pathology, suggesting that altering TDP-43''s nuclear function was sufficient to cause the disease state. Other relevant pathology in the rats included microgliosis and degenerating neuronal perikarya positive for phospho-neurofilament. The expression pattern encompassed the distribution of neuropathology of ALS, and could provide a rapid, relevant screening assay for TDP-43 variants and other disease-related proteins.     

Deregulation of TDP-43 in amyotrophic lateral sclerosis triggers nuclear factor κB-mediated pathogenic pathways

TDP-43 (TAR DNA-binding protein 43) inclusions are a hallmark of amyotrophic lateral sclerosis (ALS). In this study, we report that TDP-43 and nuclear factor κB (NF-κB) p65 messenger RNA and protein expression is higher in spinal cords in ALS patients than healthy individuals. TDP-43 interacts with and colocalizes with p65 in glial and neuronal cells from ALS patients and mice expressing wild-type and mutant TDP-43 transgenes but not in cells from healthy individuals or nontransgenic mice. TDP-43 acted as a co-activator of p65, and glial cells expressing higher amounts of TDP-43 produced more proinflammatory cytokines and neurotoxic mediators after stimulation with lipopolysaccharide or reactive oxygen species. TDP-43 overexpression in neurons also increased their vulnerability to toxic mediators. Treatment of TDP-43 mice with Withaferin A, an inhibitor of NF-κB activity, reduced denervation in the neuromuscular junction and ALS disease symptoms. We propose that TDP-43 deregulation contributes to ALS pathogenesis in part by enhancing NF-κB activation and that NF-κB may constitute a therapeutic target for the disease.     

75例结肠癌患者癌组织铜转运相关蛋白表达分析

目的探讨结肠癌组织中铜转运相关蛋白ATP7A、ATP7B和CTR1的表达与患者临床病理特征及预后的关系。方法对上海芯超生物科技有限公司提供的75例未进行任何治疗的癌组织标本组织芯片的CTR1、ATP7B和ATP7A表达面积比率进行统计分析。分析铜转运相关蛋白表达与患者临床病理特征的关系,并分析其中26例与患者生存期的关系。结果 ATP7A、ATP7B和CTR1表达水平与性别及肿瘤分期均无相关性（P〉0.05）,与年龄有关（P〈0.05）;ATP7A胞浆中位染色率为47%,胞核中位染色率为60%,胞膜不表达;ATP7B胞浆中位染色率为70%,胞核中位染色率为50%,胞膜不表达;CTR1胞浆中位染色率为52%,胞核、胞膜几乎不表达;TP7A、ATP7B和CTR1染色比率大于均数组与小于均数组之间的生存率差异无统计学意义（P〉0.05）。结论在结肠癌中铜转运相关蛋白表达水平与性别及肿瘤分期均无关,与年龄大小有关;ATP7A、ATP7B在胞浆、胞核中均有表达,在细胞膜上未见表达;ATP7A胞核表达高于胞浆,ATP7B在胞浆、胞核中均匀分布;CTR1主要在胞浆中表达,胞核、胞膜几乎不表达;CTR1、ATP7A和ATP7B在亚细胞定位的表达率与生存期无关系。     

Antibodies to human caudate nucleus neurons in Huntington''s chorea.

Antibodies reacting with neuronal cytoplasmic antigens present in normal human caudate and subthalamic nuclei were detected in 37 of 80 probands afflicted with Huntington's disease (HD). IgG antibodies were detected by immunofluorescence using frozen sections of unfixed normal human and rat brain. Specificity of IgG binding was confirmed using pepsin F(ab')2 fragments of IgG isolated from positive sera. In vitro complement fixation of IgG antibody was detected in 22 of 31 sera tested. Neuronal cytoplasmic antigens reacting with positive HD sera were diminished after trypsin or RNAase treatment of tissue sections but were not removed by DNAase, neuraminidase, EDTA, or dithiothreitol treatment. Antibody staining of neurons could be removed after absorption with isolated caudate nucleus neurons or by using perchloroacetic acid extracts of caudate nucleus. Prevalence of antibody reacting with neuronal cytoplasm was 3% in 60 normal controls and 6% among a wide variety of patients with diverse neurological disorders. However, one-third of 33 patients with Parkinson's disease showed presence of antineuronal antibody. Among patients with HD, a significant association was noted between duration of clinical disease greater than 7 yr and titers of antibody of 1:2 or greater (P less than 0.001). When 115 family members of HD probands were tested, 30% of unaffected spouses showed presence of antineuronal antibody. 23.2% of first-degree relatives at risk for developing HD was also positive (P less than 0.001). 10.5% of second-degree relatives showed presence of antineuronal antibody. These data may support an environmental or infectious factor somehow involved in the ultimate expression of HD.     

The ALS-associated proteins FUS and TDP-43 function together to affect Drosophila locomotion and life span

The fatal adult motor neuron disease amyotrophic lateral sclerosis (ALS) shares some clinical and pathological overlap with frontotemporal dementia (FTD), an early-onset neurodegenerative disorder. The RNA/DNA-binding proteins fused in sarcoma (FUS; also known as TLS) and TAR DNA binding protein-43 (TDP-43) have recently been shown to be genetically and pathologically associated with familial forms of ALS and FTD. It is currently unknown whether perturbation of these proteins results in disease through mechanisms that are independent of normal protein function or via the pathophysiological disruption of molecular processes in which they are both critical. Here, we report that Drosophila mutants in which the homolog of FUS is disrupted exhibit decreased adult viability, diminished locomotor speed, and reduced life span compared with controls. These phenotypes were fully rescued by wild-type human FUS, but not ALS-associated mutant FUS proteins. A mutant of the Drosophila homolog of TDP-43 had similar, but more severe, deficits. Through cross-rescue analysis, we demonstrated that FUS acted together with and downstream of TDP-43 in a common genetic pathway in neurons. Furthermore, we found that these proteins associated with each other in an RNA-dependent complex. Our results establish that FUS and TDP-43 function together in vivo and suggest that molecular pathways requiring the combined activities of both of these proteins may be disrupted in ALS and FTD.     

Changes in morphology of neuroblastoma cells treated with all-trans retinoic acid combined with transfer of the C-terminal region of the amyloid precursor protein

Alzheimer disease is a progressive neurodegenerative disorder that is characterized by a loss of cognitive and memory functions. Amyloid fibrils deposited in neuritic plaque is mainly β-amyloid protein (Aβ) that is derived from amyloid precursor protein (APP). The secreted form of APP, which is corresponded to N-terminal portion of APP, shows neurotrophic activities. On the other hand, Aβ and cytoplasmic domains of APP are thought to be neurotoxic. In order to investigate the effect of C-terminal fragment of APP covering Aβ and the cytoplasmic domain upon cell growth and differentiation, we established a stably transfected cell line producing the C-terminal 100 amino acid peptide of APP. The transfected clones stained positively with anti-Aβ monoclonal antibody, TB-1. The growth rate of the transfected cells was not significantly different from that of mock-transfected cells or native NB39 cells. After treatment with all-trans retinoic acid (ATRA), mock-transfected cells extended neurite processes and showed neuronal-like differentiation, while a transfected clone overexpressing C-terminal fragment did not present neuronal-like morphology. These results suggest that ATRA-induced neurite extension may be suppressed by overexpression of the C-terminal fragment of APP. J. Clin. Lab. Anal. 12:172–178, 1998.© 1998 Wiley-Liss, Inc.     

Extensive phenotype data and machine learning in prediction of mortality in acute coronary syndrome – the MADDEC study

Objective: Investigation of the clinical potential of extensive phenotype data and machine learning (ML) in the prediction of mortality in acute coronary syndrome (ACS).Methods: The value of ML and extensive clinical data was analyzed in a retrospective registry study of 9066 consecutive ACS patients (January 2007 to October 2017). Main outcome was six-month mortality. Prediction models were developed using two ML methods, logistic regression and extreme gradient boosting (xgboost). The models were fitted in training set of patients treated in 2007–2014 and 2017 (81%, n = 7344) and validated in a separate validation set of patients treated in 2015–2016 with full GRACE score data available for comparison of model accuracy (19%, n = 1722).Results: Overall, six-month mortality was 7.3% (n = 660). Several variables were found to be significantly associated with six-month mortality by both ML methods. The xgboost scored the best performance: AUC 0.890 (0.864–0.916). The AUC values for logistic regression and GRACE score were 0.867(0.837–0.897) and 0.822 (0.785–0.859), respectively. The AUC value of xgboost was better when compared to logistic regression (p = .012) and GRACE score (p < .00001).Conclusions: The use of extensive phenotype data and novel machine learning improves prediction of mortality in ACS over traditional GRACE score.

KEY MESSAGES

• The collection of extensive cardiovascular phenotype data from electronic health records as well as from data recorded by physicians can be used highly effectively in prediction of mortality after acute coronary syndrome.
• Supervised machine learning methods such as logistic regression and extreme gradient boosting using extensive phenotype data significantly outperform conventional risk assessment by the current golden standard GRACE score.
• Integration of electronic health records and the use of supervised machine learning methods can be easily applied in a single centre level to model the risk of mortality.

     

The advent of AAV9 expands applications for brain and spinal cord gene delivery

INTRODUCTION: Straightforward studies compared adeno-associated virus (AAV) serotypes to determine the most appropriate one for robust expression in the CNS. AAV9 was efficient when directly injected into the brain, but more surprisingly, AAV9 produced global expression in the brain and spinal cord after a peripheral, systemic route of administration to neonatal mice. AREAS COVERED: Topics include AAV9 gene delivery from intraparenchymal, intravenous, intrathecal and intrauterine routes of administration, and related preclinical studies and disease models. Systemic AAV9 gene transfer yields remarkably consistent neuronal expression, though only in early development. AAV9 is versatile to study neuropathological proteins: microtubule-associated protein tau and transactive response DNA-binding protein 43 kDa (TDP-43). EXPERT OPINION: AAV9 will be more widely used based on current data, although other natural serotypes and recombineered vectors may also support or improve upon wide-scale expression. A peripheral-to-central gene delivery that can affect the entire CNS without having to inject the CNS is promising for basic functional experiments, and potentially for gene therapy. Systemic or intra-cerebrospinal fluid routes of AAV9 administration should be considered for spinal muscular atrophy, lysosomal storage diseases and amyotrophic lateral sclerosis, if more neuronal expression can be achieved in adults, or if glial expression can be exploited.     

Cytoplasmic CUG RNA Foci Are Insufficient to Elicit Key DM1 Features

The genetic basis of myotonic dystrophy type I (DM1) is the expansion of a CTG tract located in the 3′ untranslated region of DMPK. Expression of mutant RNAs encoding expanded CUG repeats plays a central role in the development of cardiac disease in DM1. Expanded CUG tracts form both nuclear and cytoplasmic aggregates, yet the relative significance of such aggregates in eliciting DM1 pathology is unclear. To test the pathophysiology of CUG repeat encoding RNAs, we developed and analyzed mice with cardiac-specific expression of a beta-galactosidase cassette in which a (CTG)₄₀₀ repeat tract was positioned 3′ of the termination codon and 5′ of the bovine growth hormone polyadenylation signal. In these animals CUG aggregates form exclusively in the cytoplasm of cardiac cells. A key pathological consequence of expanded CUG repeat RNA expression in DM1 is aberrant RNA splicing. Abnormal splicing results from the functional inactivation of MBNL1, which is hypothesized to occur due to MBNL1 sequestration in CUG foci or from elevated levels of CUG-BP1. We therefore tested the ability of cytoplasmic CUG foci to elicit these changes. Aggregation of CUG RNAs within the cytoplasm results both in Mbnl1 sequestration and in approximately a two fold increase in both nuclear and cytoplasmic Cug-bp1 levels. Significantly, despite these changes RNA splice defects were not observed and functional analysis revealed only subtle cardiac dysfunction, characterized by conduction defects that primarily manifest under anesthesia. Using a human myoblast culture system we show that this transgene, when expressed at similar levels to a second transgene, which encodes expanded CTG tracts and facilitates both nuclear focus formation and aberrant splicing, does not elicit aberrant splicing. Thus the lack of toxicity of cytoplasmic CUG foci does not appear to be a consequence of low expression levels. Our results therefore demonstrate that the cellular location of CUG RNA aggregates is an important variable that influences toxicity and support the hypothesis that small molecules that increase the rate of transport of the mutant DMPK RNA from the nucleus into the cytoplasm may significantly improve DM1 pathology.     

胃肠道黏膜相关淋巴组织淋巴瘤BCL-10的表达

目的　探讨BCL 10蛋白在胃肠道黏膜相关淋巴组织淋巴瘤 (MALToma)中表达的意义。方法　应用CD2 0、CD79a、CD3、CD4 5RO、CD2 3、CD5、CD10单克隆抗体对 4 3例胃肠道MALToma进行检测 ,其中 2 5例为经典MALToma(MALToma LG) ,临床为惰性 ;18例为伴有少数大细胞转化的MALToma(TransformedMALToma)。用SABC免疫组化的方法检测瘤细胞BCL 10蛋白的表达。结果　在 2 5例惰性MALToma标本中 10例为BCL 10核阳性 ,1例为BCL 10核与浆同时阳性 ,3例为瘤细胞浆阳性 ,11例为阴性 ;在 18例转化的MALToma中 7例为BCL 10核阳性 ,1例为BCL 10核与浆同时阳性 ,2例为瘤细胞浆阳性 ,8例为阴性。BCL 10核阳性在胃肠道MALToma中的出现率较高 ,达 4 4 .2 %。结论　BCL 10在胃肠道MALToma中发生率较高 ,该高发生率与肿瘤的发生或演进可能有一定的相关性     

Chaperone Nanobodies Protect Gelsolin Against MT1-MMP Degradation and Alleviate Amyloid Burden in the Gelsolin Amyloidosis Mouse Model

Gelsolin amyloidosis is an autosomal dominant incurable disease caused by a point mutation in the GSN gene (G654A/T), specifically affecting secreted plasma gelsolin. Incorrect folding of the mutant (D187N/Y) second gelsolin domain leads to a pathological proteolytic cascade. D187N/Y gelsolin is first cleaved by furin in the trans-Golgi network, generating a 68 kDa fragment (C68). Upon secretion, C68 is cleaved by MT1-MMP-like proteases in the extracellular matrix, releasing 8 kDa and 5 kDa amyloidogenic peptides which aggregate in multiple tissues and cause disease-associated symptoms. We developed nanobodies that recognize the C68 fragment, but not native wild type gelsolin, and used these as molecular chaperones to mitigate gelsolin amyloid buildup in a mouse model that recapitulates the proteolytic cascade. We identified gelsolin nanobodies that potently reduce C68 proteolysis by MT1-MMP in vitro. Converting these nanobodies into an albumin-binding format drastically increased their serum half-life in mice, rendering them suitable for intraperitoneal injection. A 12-week treatment schedule of heterozygote D187N gelsolin transgenic mice with recombinant bispecific gelsolin-albumin nanobody significantly decreased gelsolin buildup in the endomysium and concomitantly improved muscle contractile properties. These findings demonstrate that nanobodies may be of considerable value in the treatment of gelsolin amyloidosis and related diseases.     

Should pulse oximetry be included in GPs’ assessment of patients with obstructive lung disease?

Objective: To explore the associations between decreased pulse oximetry values (SpO₂) and clinical, laboratory, and demographic variables in general practice patients diagnosed with asthma or chronic obstructive pulmonary disease (COPD), including those with both COPD and asthma in combination.Design/setting: A cross-sectional study in seven Norwegian general practices of patients aged 40 years or over who were diagnosed by their general practitioner (GP) with asthma and/or COPD. The patients were examined during a stable phase of their disease. Patients diagnosed with COPD (including those with combined COPD/asthma) and those diagnosed with asthma only were analysed separately.Main outcome measures: Decreased SpO₂ values (≤ 95% and ≤ 92%).Results: Of 372 patients included (mean age 61.5 years, 62% women), 82 (22.0%) had SpO₂ ≤ 95%, of which 11 had SpO₂ ≤ 92%. In both asthma and COPD patients, SpO₂ ≤ 95% was significantly associated with reduced lung function (spirometry), a diagnosis of coronary heart disease and older age (≥ 65 years). In the COPD group, haemoglobin above normal was associated with SpO₂ ≤ 95%. These associations were confirmed by multivariable logistic regression, where FEV₁% predicted < 50 was the strongest predictor of SpO₂ ≤ 95% (odds ratio 6.8, 95% confidence interval 2.8–16.4).Conclusion. Pulse oximetry represents a useful diagnostic adjunct for assessing the severity of obstructive pulmonary disease. Decreased pulse oximetry values in stable-phase patients with asthma and/or COPD should prompt the GP to consider revising the diagnosis and treatment and to look for co-morbidities.

Key Points

• Despite its common use in general practice, the diagnostic benefits of pulse oximetry remain to be established.
• Decreased pulse oximetry values are associated with both reduced lung function (spirometry) and with a diagnosis of coronary heart disease.
• Decreased pulse oximetry values may reflect suboptimal treatment and/or undiagnosed comorbidity.
• Pulse oximetry may therefore be a useful measure in the follow-up of asthma and COPD patients in general practice.

     

The advent of AAV9 expands applications for brain and spinal cord gene delivery

Introduction: Straightforward studies compared adeno-associated virus (AAV) serotypes to determine the most appropriate one for robust expression in the CNS. AAV9 was efficient when directly injected into the brain, but more surprisingly, AAV9 produced global expression in the brain and spinal cord after a peripheral, systemic route of administration to neonatal mice.

Areas covered: Topics include AAV9 gene delivery from intraparenchymal, intravenous, intrathecal and intrauterine routes of administration, and related preclinical studies and disease models. Systemic AAV9 gene transfer yields remarkably consistent neuronal expression, though only in early development. AAV9 is versatile to study neuropathological proteins: microtubule-associated protein tau and transactive response DNA-binding protein 43 kDa (TDP-43).

Expert opinion: AAV9 will be more widely used based on current data, although other natural serotypes and recombineered vectors may also support or improve upon wide-scale expression. A peripheral-to-central gene delivery that can affect the entire CNS without having to inject the CNS is promising for basic functional experiments, and potentially for gene therapy. Systemic or intra-cerebrospinal fluid routes of AAV9 administration should be considered for spinal muscular atrophy, lysosomal storage diseases and amyotrophic lateral sclerosis, if more neuronal expression can be achieved in adults, or if glial expression can be exploited.