Rare V203I mutation in the PRNP gene of a Chinese patient with Creutzfeldt–Jakob disease

Here, we report a Chinese case of Creutzfeldt–Jakob disease (CJD) with a rare mutation in the prion protein gene (PRNP) leading to an exchange of amino acid from valine (Val) to isoleucine (I) at codon 203 (V203I). The 80-y-old male presented with sudden memory loss, rapid loss of vocabulary, inattention and slow responses, accompanied by dizziness, blurred vision and ataxia. Two weeks after admission, he exhibited tremor, myoclonus and bilateral Babinski signs. At the end of the clinical course, he developed severe akinetic mutism. The cerebrospinal fluid (CSF) was positive for 14-3-3 protein. Increased bilateral signal intensity in the frontal and parietal lobes was seen on diffusion-weighted imaging (DWI); periodic activity was recorded on an electroencephalogram (EEG). There was no family history of similar symptoms. The total clinical course was approximately two months.     

Creutzfeldt-Jakob Disease with a prion protein gene codon 180 mutation presenting asymmetric cortical high-intensity on magnetic resonance imaging

ABSTRACT. Here we report a genetically confirmed case of Creutzfeldt-Jakob disease with a prion protein gene codon 180 mutation presenting atypical magnetic resonance imaging findings. The present case exhibited an acute onset and lateralized neurologic signs, and progressive cognitive impairment. No myoclonus or periodic synchronous discharges on electroencephalography were observed. Diffusion-weighted images revealed areas of high signal intensity in the right frontal and temporal cortices at onset that extended to the whole cortex and basal ganglia of the right cerebral hemisphere at 3 months. Although the cerebrospinal fluid (CSF) was initially negative for neuron specific enolase, tau protein, 14–3–3 protein, and abnormal prion protein, the CSF was positive for these brain-derived proteins at 3 months after onset.     

Creutzfeldt-Jakob Disease with a prion protein gene codon 180 mutation presenting asymmetric cortical high-intensity on magnetic resonance imaging

ABSTRACT. Here we report a genetically confirmed case of Creutzfeldt-Jakob disease with a prion protein gene codon 180 mutation presenting atypical magnetic resonance imaging findings. The present case exhibited an acute onset and lateralized neurologic signs, and progressive cognitive impairment. No myoclonus or periodic synchronous discharges on electroencephalography were observed. Diffusion-weighted images revealed areas of high signal intensity in the right frontal and temporal cortices at onset that extended to the whole cortex and basal ganglia of the right cerebral hemisphere at 3 months. Although the cerebrospinal fluid (CSF) was initially negative for neuron specific enolase, tau protein, 14–3–3 protein, and abnormal prion protein, the CSF was positive for these brain-derived proteins at 3 months after onset.     

Relationships between Clinicopathological Features and Cerebrospinal Fluid Biomarkers in Japanese Patients with Genetic Prion Diseases

A national system for surveillance of prion diseases (PrDs) was established in Japan in April 1999. Here, we analyzed the relationships among prion protein gene (PRNP) mutations and the clinical features, cerebrospinal fluid (CSF) markers, and pathological characteristics of the major genotypes of genetic PrDs (gPrDs). We retrospectively analyzed age at onset and disease duration; the concentrations and incidences of 14-3-3 protein, tau protein, and abnormal prion protein (PrP^Sc) in the CSF of 309 gPrD patients with P102L, P105L, E200K, V180I, or M232R mutations; and brain pathology in 32 autopsied patients. Three clinical phenotypes were seen: rapidly progressive Creutzfeldt-Jakob disease (CJD), which included 100% of E200K cases, 70% of M232R, and 21% of P102L; slowly progressive CJD, which included 100% of V180I and 30% of M232R; and Gerstmann-Sträussler-Scheinker disease, which included 100% of P105L and 79% of P102L. PrP^Sc was detected in the CSF of more than 80% of patients with E200K, M232R, or P102L mutations but in only 39% of patients with V180I. V180I was accompanied by weak PrP immunoreactivity in the brain. Patients negative for PrP^Sc in the CSF were older at disease onset than positive patients. Patients with mutations associated with high 14-3-3 protein levels in the CSF typically had synaptic deposition of PrP in the brain and a rapid course of disease. The presence of small PrP protein fragments in brain homogenates was not correlated with other clinicopathological features. Positivity for PrP^Sc in the CSF may reflect the pathological process before or at disease onset, or abnormality in the secretion or metabolism of PrP^Sc. The amount of 14-3-3 protein in the CSF likely indicates the severity of the pathological process and accompanying neuronal damage. These characteristic features of the CSF in cases of gPrD will likely facilitate accurate diagnosis and clinicopathological study of the various disease subtypes.     

First case of V180I rare mutation in a Brazilian patient with Creutzfeldt-Jakob disease

Here, we report the first case of V180I rare mutation in a Brazilian woman whose clinical condition started with memory impairment for recent events and insomnia with 2 months of evolution, without any other alterations in neurological examination. Both the electroencephalogram (EEG) and the routine biochemical examination of cerebrospinal fluid (CSF) were normal. CSF 14-3-3 protein search was positive. Magnetic resonance imaging (MRI) of the encephalon showed findings suggestive of Creutzfeldt-Jakob disease, confirmed by sequencing of PRNP gene that reveal V180I mutation also homozygosity for methionine at codon 129 (M129M).     

Functional Analysis LRP6 Novel Mutations in Patients with Coronary Artery Disease

Background

Genetic architecture of coronary artery disease (CAD) is still to be defined. Since low density lipoprotein receptor-related protein 6 (LRP6) gene play critical roles in Wnt signal transduction which are important for vascular development and endodermis specification, we therefore resequenced it to search for mutations in CAD patients.

Methods

We systemically sequenced all the exons and promoter region of LRP6 gene in a sample of 380 early onset CAD patients and 380 control subjects in Chinese.

Results

In total, we identified 5 patient-specific mutations including K82N (two patients), S488Y (one patient), P1066T (two patients), P1206H (two patients) and I1264V (one patient) All these mutations located at the extracellular domain of LRP6 gene. In vitro functional analysis of patient-specific mutations demonstrated that these mutations resulted in a significant reduction in both protein level transporting to cell membrane and downstream Wnt signal activity. Furthermore, we found that LRP6 novel mutations attenuated proliferation and migration of human umbilical vein endothelial cells (HUVECs) when compared with wild type (WT) LRP6.

Conclusion

Our results demonstrated that these loss-of-function variants might contribute to disease liability in a subset of CAD and defects in Wnt signal activation might be important contributing factors for the onset of CAD.     

Pathological progression of genetic Creutzfeldt–Jakob disease with a PrP V180I mutation

In comparison to sporadic Creutzfeldt–Jakob disease (sCJD) with MM1-type and MM2- cortical (MM2C)-type, genetic CJD with a prion protein gene V180I mutation (V180I gCJD) is clinically characterized by onset at an older age, slower progress, and the absence of visual disturbances or cerebellar symptoms. In terms of pathological characteristics, gliosis and neuronal loss are generally milder in degree, and characteristic spongiform change can be observed at both the early and advanced stages. However, little is known on the progress of spongiform change over time or its mechanisms. In this study, to elucidate the pathological course of V180I gCJD, statistical analysis of the size and dispersion of the major diameters of vacuoles in six V180I gCJD cases was performed, with five MM1-type sCJD and MM2C-type sCJD cases as controls. As a result, V180I gCJD showed no significant difference in vacuolar diameter regardless of disease duration. In addition, the dispersion of the major diameters of vacuoles in V180I gCJD was larger than that in the MM1-type, which was smaller than that in the MM2C-type. We speculated that the absence of difference in the size of the vacuoles regardless of disease duration suggests that tissue rarefaction does not result from the expansion of vacuole size and increase in number of vacuoles in V180Ig CJD. These features were considered to be significant pathological findings of V180I gCJD.     

Creutzfeldt-Jakob disease associated with a V203I homozygous mutation in the prion protein gene

We report a Japanese patient with Creutzfeldt-Jakob disease (CJD) with a V203I homozygous mutation of the prion protein gene (PRNP). A 73-year-old woman developed rapidly progressive gait disturbance and cognitive dysfunction. Four months after the onset, she entered a state of an akinetic mutism. Gene analysis revealed a homozygous V203I mutation in the PRNP. Familial CJD with a V203I mutation is rare, and all previously reported cases had a heterozygous mutation showing manifestations similar to those of typical sporadic CJD. Although genetic prion diseases with homozygous PRNP mutations often present with an earlier onset and more rapid clinical course than those with heterozygous mutations, no difference was found in clinical phenotype between our homozygous case and reported heterozygous cases.     

Rare E196A mutation in PRNP gene of 3 Chinese patients with Creutzfeldt-Jacob disease

Inherited prion diseases are characterized by mutations in the PRNP gene, which account for 5–15% of human prion diseases. Here we reported 3 Chinese genetic Creutzfeldt-Jacob disease cases (gCJD) with a rare mutation in PRNP leading to an exchange of amino acid from glutamic acid (E) to alanine (A) at codon 196 (E196A). All three patients were Han Chinese without any sibship among them. They showed various unspecific symptoms at onset and displayed typical clinical manifestations of sporadic CJD with progress of disease. The same time, 2 cases showed psychotic symptoms during the clinical courses. 14-3-3 proteins were positive in cerebrospinal fluid (CSF) and special abnormality were detected in MRI of all the cases. The polymorphism of codon 129 was methionin homozygote and that of codon 219 was glutamate homozygote in all 3 patients. The disease durations of the 3 cases varied from 10 to 22 months and no disease associated family history was figured out in all the cases.     

A Chinese patient of P102L Gerstmann-Sträussler-Scheinker disease contains three other disease-associated mutations in SYNE1

Gerstmann-Sträussler-Scheinker disease (GSS) with the P102L mutation in PRNP gene is characterized with progressive cerebellar dysfunction clinically and PrP^Sc plaques neurologically. Due to the cerebellar ataxia in the early stage, GSS P102L is often misdiagnosed as other neurodegenerative disorders. We presented here a 49-year-old female patient with proven P102L PRNP mutation, and three heterologous mutations in hereditary ataxias associated gene SYNE1, including p.V3643L, p.M3376V and p.T2860A. The patient appeared progressive unsteady gait in early stage and developed the Creutzfeldt-Jacob disease (CJD) – associated clinical manifestations, including progressive dementia, myoclonus, pyramidal and extrapyramidal signs. She is still alive but with akinetic mutism 21 months after onset. Observation of intense signal changes in cortical regions (cortical ribboning) in diffusion weighted imaging (DWI) MRI scanning and positive protein 14-3-3 in cerebrospinal fluid (CSF) proposed the diagnosis of sporadic CJD. The final diagnosis of P102L GSS was made after PRNP sequencing.     

Connective tissue growth factor and cardiac fibrosis after myocardial infarction.

The temporal and spatial expression of transforming growth factor (TGF)-beta(1) and connective tissue growth factor (CTGF) was assessed in the left ventricle of a myocardial infarction (MI) model of injury with and without angiotensin-converting enzyme (ACE) inhibition. Coronary artery ligated rats were killed 1, 3, 7, 28, and 180 days after MI. TGF-beta(1), CTGF, and procollagen alpha1(I) mRNA were localized by in situ hybridization, and TGF-beta(1) and CTGF protein levels by immunohistochemistry. Collagen protein was measured using picrosirius red staining. In a separate group, rats were treated for 6 months with an ACE inhibitor. There were temporal and regional differences in the expression of TGF-beta(1), CTGF, and collagen after MI. Procollagen alpha1(I) mRNA expression increased in the border zone and scar peaking 1 week after MI, whereas collagen protein increased in all areas of the heart over the 180 days. Expression of TGF-beta(1) mRNA and protein showed major increases in the border zone and scar peaking 1 week after MI. The major increases in CTGF mRNA and protein occurred in the viable myocardium at 180 days after MI. Long-term ACE inhibition reduced left ventricular mass and decreased fibrosis in the viable myocardium, but had no effect on cardiac TGF-beta(1) or CTGF. TGF-beta(1) is involved in the initial, acute phase of inflammation and repair after MI, whereas CTGF is involved in the ongoing fibrosis of the heart. The antifibrotic benefits of captopril are not mediated through a reduction in CTGF.     

3-(Trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine: Synthesis and chemical characterization of a heterobifunctional carbene-generating crosslinking reagent

A new hydrophobic heterobifunctional photocrosslinking reagent 3-(trifluoromethyl)-3-(m-isothiocyanophenyl)diazirine (TRIMID), a carbene precursor, and its radioiodinated analogue [¹²⁵I]TRIMID, have been synthesized and chemically characterized. The reagents were applied for membrane protein modification in human erythrocyte membranes and purple membranes fromHalobacterium halobium. Covalent labeling of the anion transport protein (band 3) via the isothiocyanate function was confirmed. Radiolabeled TRIMID was detected in at least two thermolysin-generated transmembrane fragments of the anion transport protein, and half-maximal inhibition of the erythrocyte anion transport activity was attained with 2.2 mM reagent. In bacteriorhodopsin (BR), a common binding site for the monofunctional phenylisothiocyanate and the bifunctional crosslinking reagent was identified: preincubation of purple membranes with TRIMID suppressed phenylisothio-[¹⁴C]-cyanate binding to BR. [¹²⁵I]TRIMID was recovered in V-1, the N-terminal segment of BR, which includes the phenylisothiocyanate binding site Lys-41. Light-induced intramolecular crosslinking of band 3-derived thermolytic fragments was not observed, although the carbene was generatedin situ and photocrosslinking of the protease V8 fragments of BR was not detected. Chemical and physicochemical characteristics of the new reagent are discussed with regard to limitations imposed for photoinduced site-directed crosslink formation.     

O-GlcNAc transferase promotes the nuclear localization of the focal adhesion–associated protein Zyxin to regulate UV-induced cell death

Zyxin is a zinc-binding phosphoprotein known to regulate cell migration, adhesion, and cell survival. Zyxin also plays a role in signal transduction between focal adhesions and the nuclear compartment. However, the mechanism of Zyxin shuttling to nucleus is still unclear. Here, we identify that the GlcNAc transferase (O-linked GlcNAc [O-GlcNAc] transferase) can O-GlcNAcylate Zyxin and regulate its nuclear localization. We show that O-GlcNAc transferase O-GlcNAcylates Zyxin at two residues, serine 169 (Ser-169) and Ser-246. In addition, O-GlcNAcylation of Ser-169, but not Ser-246, enhances its interaction with 14-3-3γ, which is a phosphoserine/threonine-binding protein and is reported to bind with phosphorylated Zyxin. Furthermore, we found that 14-3-3γ could promote the nuclear localization of Zyxin after Ser-169 O-GlcNAcylation by affecting the function of the N-terminal nuclear export signal sequence; functionally, UV treatment increases the O-GlcNAcylation of Zyxin, which may enhance the nuclear location of Zyxin. Finally, Zyxin in the nucleus maintains homeodomain-interacting protein kinase 2 stability and promotes UV-induced cell death. In conclusion, we uncover that the nuclear localization of Zyxin can be regulated by its O-GlcNAcylation, and that this protein may regulate UV-induced cell death.     

Identification and functional analysis of a new putative caveolin-3 variant found in a patient with sudden unexplained death

Background

Sudden cardiac death (SCD) is the clinical outcome of a lethal arrhythmia that can develop on the background of unrecognized channelopathies or cardiomyopathies. Several susceptibility genes have been identified for the congenital forms of these cardiac diseases, including caveolin-3 (Cav-3) gene. In the heart Cav-3 is the main component of caveolae, plasma membrane domains that regulate multiple cellular processes highly relevant for cardiac excitability, such as trafficking, calcium homeostasis, signal transduction and cellular response to injury. Here we characterized a new putative Cav-3 variant, Cav-3 V82I, found in a patient with SCD.

Results

In heterologous systems Cav-3 V82I was expressed at significantly higher level than Cav-3 WT and accumulated within the cells. Cells expressing Cav-3 V82I exhibited a decreased activation of extracellular-signal-regulated kinases (ERKs) and were more vulnerable to sub-lethal osmotic stress.

Conclusion

Considering that abnormal loss of myocytes can play a mechanistic role in lethal cardiac diseases, we suggest that the detrimental effect of Cav-3 V82I variant on cell viability may participate in determining the susceptibility to cardiac death.     

14-3-3 Protein, Total Tau and Phosphorylated Tau in Cerebrospinal Fluid of Patients with Creutzfeldt-Jakob Disease and Neurodegenerative Disease in Japan

1.Sporadic Creutzfeldt-Jakob disease (CJD) is a rapidly progressive and fatal disease. Patients with CJD usually become akinetic mutism within approximately 6 months. In addition, clinical signs and symptoms at early stage of sporadic CJD may not be easy to distinguish from other neurodegenerative diseases by neurological findings. However, diagnostic biochemical parameters including 14-3-3 protein, S100, neuron-specific enorase in cerebrospinal fluid (CSF) have been used as diagnostic markers, elevated titers of these markers can also be observed in CSF in other neurodegenerative diseases. Therefore, we examined other biochemical markers to discriminate CJD from other neurodegenerative diseases in CSF. 2.We analyzed CSF samples derived from 100 patients with various neurodegenerative disorders by Western blot of 14-3-3 protein, quantification of total tau (t-tau) protein, and phosphorylated tau (p-tau) protein. All patients with CJD in this study showed positive 14-3-3 protein and elevated t-tau protein (>1000 pg/mL) in CSF. We also detected positive 14-3-3 protein bands in two patients in non-CJD group (patients with dementia of Alzheimer's type; DAT) and also detected elevated t-tau protein in three patients in non-CJD group. Elevated t-tau protein levels were observed in two patients with DAT and in one patient with cerevrovascular disease in acute phase. 3.To distinguish patients with CJD from non-CJD patients with elevated t-tau protein in CSF, we compared the ratio of p-tau and t-tau proteins. The p-/t-tau ratio was dramatically and significantly higher in DAT patients rather than in CJD patients. 4.Therefore, we concluded that the assay of t-tau protein may be useful as 1st screening and the ratio of p-tau protein/t-tau protein would be useful as 2nd screening to discriminate CJD from other neurodegenerative diseases.     

Preserved synaptic vesicle recycling in hippocampal neurons in a mouse Alzheimer's disease model

A recently described triple-transgenic mouse model (3xTg, PS1(M146V), APP(Swe), and tau(P301L)) develops a neuropathology similar to the brains of Alzheimer's disease patients including progressive deposits of plaques and tangles [Neuron 39 (2003) 409]. These mice also show age-related deficits in hippocampal synaptic plasticity that occurs before the development of plaques and tangles. Here we report unchanged synaptic vesicle recycling, as measured by FM1-43 release, in the hippocampal neurons of the 3xTg mice. Expression levels of presynaptic protein synaptophysin and of proteins involved in synaptic vesicle recycling including AP180, dynamin I, and synaptotagmin I also remain unaffected. These data suggest that the synaptic deficits observed in the 3xTg neurons may not arise from the preserved synaptic vesicle recycling.     

Isoform pattern of 14-3-3 proteins in the cerebrospinal fluid of patients with Creutzfeldt-Jakob disease

Two-dimensional polyacrylamide gel electrophoresis of CSF has been used in the diagnosis of Creutzfeldt-Jakob disease (CJD). One of the two diagnostic protein spots was identified as isoform(s) of the 14-3-3 family of abundant brain proteins. This has led to the development of one-dimensional 14-3-3 sodium dodecyl sulfate polyacrylamide gel electrophoresis immunoblot, which is currently used to support the diagnosis of CJD. In the present study employing western blot analysis, we have identified the panel of 14-3-3 isoforms that appear in the CSF of 10 patients with CJD compared with 10 patients with other dementias. The results clearly show that the 14-3-3 isoforms beta, gamma, epsilon, and eta are present in the CSF of patients with CJD and can be used to differentiate other dementias. 14-3-3eta also gave a baseline signal in all patients with other dementias, including six patients with Alzheimer's disease. The presence of 14-3-3eta in the CSF of a patient with herpes simplex encephalitis was particularly noteworthy. This study has determined that isoform-specific 14-3-3 antibodies against beta, gamma, and epsilon should be considered for the neurochemical differentiation of CJD from other neurodegenerative diseases.     

Nuclear export signal in CDC25B

CDC25B is a dual-specificity phosphatase that activates CDK1/cyclin B. The nuclear exclusion of CDC25B is controlled by the binding of 14-3-3 to the nuclear export signal (NES) of CDC25B, which was reported to be amino acids H28 to L40 in the N-terminal region of CDC25B. In studying the subcellular localization of CDC25B, we found a functional NES at V52 to L65, the sequence of which is VTTLTQTMHDLAGL, where bold letters are leucine or hydrophobic amino acids frequently seen in an NES. The deletion of this NES sequence caused the mutant protein to locate exclusively in nuclei, while NES-fused GFP was detected in the cytoplasm. Moreover, the introduction of point mutations at some of the critical amino acids impaired cytoplasmic localization. Treatment with leptomycin B, a potent inhibitor of CRM1/exportin1, disrupted the cytoplasmic localization of both Flag-tagged CDC25B and NES-fused GFP. From these results, we concluded that the sequence we found is a bona fide NES of CDC25B.     

A multimeric membrane protein reveals 14-3-3 isoform specificity in forward transport in yeast

Arginine (Arg)-based endoplasmic reticulum (ER) localization signals are sorting motifs involved in the quality control of multimeric membrane proteins. They are distinct from other ER localization signals like the C-terminal di-lysine [-K(X)KXX] signal. The Pmp2p isoproteolipid, a type I yeast membrane protein, reports faithfully on the activity of sorting signals when fused to a tail containing either an Arg-based motif or a -KKXX signal. This reporter reveals that the Arg-based ER localization signals from mammalian Kir6.2 and GB1 proteins are functional in yeast. Thus, the machinery involved in recognition of Arg-based signals is evolutionarily conserved. Multimeric presentation of the Arg-based signal from Kir6.2 on Pmp2p results in forward transport, which requires 14-3-3 proteins encoded in yeast by BMH1 and BMH2 in two isoforms. Comparison of a strain without any 14-3-3 proteins (Deltabmh2) and the individual Deltabmh1 or Deltabmh2 shows that the role of 14-3-3 in the trafficking of this multimeric Pmp2p reporter is isoform-specific. Efficient forward transport requires the presence of Bmh1p. The specific role of Bmh1p is not due to differences in abundance or affinity between the isoforms. Our results imply that 14-3-3 proteins mediate forward transport by a mechanism distinct from simple masking of the Arg-based signal.