An autopsied case of MM1-type sporadic Creutzfeldt-Jakob disease with pathology of Wernicke encephalopathy

An 83-year-old Japanese man presented with gait disturbance followed by rapidly-progressive cognitive impairment. Magnetic resonance diffusion-weighted images showed extensive hyperintense regions in the cerebral cortex. Four weeks after symptom onset, myoclonus appeared, and the patient developed difficulty swallowing; intravenous peripheral continuous infusions without vitamin supplementation were administered during the last two months of the patient’s life. The patient reached the akinetic mutism state and died 12 weeks after symptom onset due to sepsis. The brain weighed 940 g and showed general cerebral atrophy. Extensive spongiform change were observed in the cerebral cortex, striatum, thalamus, and cerebellar cortex, but gliosis was generally mild. Numerous newly-developed hemorrhage foci were observed in the mammillary body, the areas adjacent to the third and fourth ventricles, and the periaqueduct of the midbrain; however, proliferation of capillaries and endothelium and collections of macrophages were relatively inconspicuous. These findings suggested comorbidity with the acute stage of Wernicke encephalopathy (WE). Immunostaining showed extensive diffuse synaptic-type prion protein deposition in the gray matter. According to the neuropathological, genetic, and molecular findings, the present case was finally diagnosed as MM1-type sporadic Creutzfeldt-Jakob disease (CJD) with WE. We should remain alert to the diagnosis of WE when CJD is suspected, and it is necessary to consider the complications of both diseases. This report emphasizes the importance of pathological investigations for the diagnosis of CJD, WE, and the coexistence of both.     

A de novo deafwaddler mutation of Pmca2 arising in ES cells and hitchhiking with a targeted modification of the Pparg gene

We observed severe ataxia in mice homozygous for modification of the Pparg locus. Genetic analysis and nucleotide sequencing revealed that ataxia is caused by a T692K substitution in plasma membrane calcium ATPase 2 (Pmca2), which is tightly linked to Pparg, but not by modified PPARγ itself. We traced this mutation and found that it arose spontaneously during clonal expansion of the targeted embryonic stem (ES) cells. Consistent with the deafwaddler phenotype in other Pmca2 mutants, homozygous T692K Pmca2 mutants exhibit severe balance disorder, impaired neurologic reflexes, and motor coordination, and have profound hearing loss. Heterozygous mutants have normal movement and motor function but are severely deficient in hearing. Our findings represent a cautionary example since, although rare, spontaneous mutations do arise in ES cells during culture and hitchhike onto the targeted gene mutation. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. This article contains a supplementary video.     

A Slow Maturation of a Cysteine Protease with a Granulin Domain in the Vacuoles of Senescing Arabidopsis Leaves

Arabidopsis RD21 is a cysteine protease of the papain family. Unlike other members of the papain family, RD21 has a C-terminal extension sequence composed of two domains, a 2-kD proline-rich domain and a 10-kD domain homologous to animal epithelin/granulin family proteins. The RD21 protein was accumulated as 38- and 33-kD proteins in Arabidopsis leaves. An immunoblot showed that the 38-kD protein had the granulin domain, whereas the 33-kD protein did not. A pulse-chase experiment with Bright-Yellow 2 transformant cells expressing RD21 showed that RD21 was synthesized as a 57-kD precursor and was then slowly processed to make the 33-kD mature protein via the 38-kD intermediate. After a 12-h chase, the 38-kD intermediate was still detected in the cells. These results indicate that the N-terminal propeptide was first removed from the 57-kD precursor, and the C-terminal granulin domain was then slowly removed to yield the 33-kD mature protein. Subcellular fractionation of the Bright-Yellow 2 transformant showed that the intermediate and mature forms of RD21 were localized in the vacuoles. Under the acidic conditions of the vacuolar interior, the intermediate was found to be easily aggregated. The intermediate and the mature protein were accumulated in association with leaf senescence. Taken together, these results indicate that the intermediate of RD21 was accumulated in the vacuoles as an aggregate, and then slowly matured to make a soluble protease by removing the granulin domain during leaf senescence.     

AtVAM3 is required for normal specification of idioblasts, myrosin cells

Myrosin cells in Capparales plants are idioblasts that accumulate thioglucoside glucohydrolase (TGG, also called myrosinase), which hydrolyzes glucosinolates to produce toxic compounds for repelling pests. Here, we show that AtVAM3 is involved in development of myrosin cells. It has been shown that yeast VAM3 is a Q(a)-SNARE that is involved in vesicle transport of vacuolar proteins and vacuolar assembly. We found that two Arabidopsis atvam3 alleles, atvam3-3 and atvam3-4/ssm, accumulate large amounts of TGG1 and TGG2 that are enzymatically active. An immunogold analysis revealed that TGGs were specifically localized in the vacuole of myrosin cells in atvam3 mutants. This result indicates that TGGs are normally transported to vacuoles in these mutants and that AtVAM3 is not essential for vacuolar transport of the proteins. We developed a staining method with Coomassie brilliant blue that detects myrosin cells in whole leaves by their high TGG content. This method showed that atvam3 leaves have a larger number of myrosin cells than do wild-type leaves. Myrosin cells were scattered along leaf veins in wild-type leaves, while they were abnormally distributed in atvam3 leaves. The mutants developed a network of myrosin cells throughout the leaves: myrosin cells were not only distributed continuously along leaf veins, but were also observed independent of leaf veins. The excess of myrosin cells in atvam3 mutants might be responsible for the abnormal abundance of TGGs and the reduction of elongation of inflorescence stems and leaves in these mutants. Our results suggest that AtVAM3 has a plant-specific function in development of myrosin cells.     

Retromer Contributes to Immunity-Associated Cell Death in Arabidopsis

Membrane trafficking is required during plant immune responses, but its contribution to the hypersensitive response (HR), a form of programmed cell death (PCD) associated with effector-triggered immunity, is not well understood. HR is induced by nucleotide binding-leucine-rich repeat (NB-LRR) immune receptors and can involve vacuole-mediated processes, including autophagy. We previously isolated lazarus (laz) suppressors of autoimmunity-triggered PCD in the Arabidopsis thaliana mutant accelerated cell death11 (acd11) and demonstrated that the cell death phenotype is due to ectopic activation of the LAZ5 NB-LRR. We report here that laz4 is mutated in one of three VACUOLAR PROTEIN SORTING35 (VPS35) genes. We verify that LAZ4/VPS35B is part of the retromer complex, which functions in endosomal protein sorting and vacuolar trafficking. We show that VPS35B acts in an endosomal trafficking pathway and plays a role in LAZ5-dependent acd11 cell death. Furthermore, we find that VPS35 homologs contribute to certain forms of NB-LRR protein-mediated autoimmunity as well as pathogen-triggered HR. Finally, we demonstrate that retromer deficiency causes defects in late endocytic/lytic compartments and impairs autophagy-associated vacuolar processes. Our findings indicate important roles of retromer-mediated trafficking during the HR; these may include endosomal sorting of immune components and targeting of vacuolar cargo.     

Purification, characterization and reconstitution into membranes of the oligomeric c-subunit ring of thermophilic F(o)F(1)-ATP synthase expressed in Escherichia coli

F(o)F(1)-ATP synthase catalyzes ATP synthesis coupled with proton-translocation across the membrane. The membrane-embedded F(o) portion is responsible for the H(+) translocation coupled with rotation of the oligomeric c-subunit ring, which induces rotation of the γ subunit of F(1). For solid-state NMR measurements, F(o)F(1) of thermophilic Bacillus PS3 (TF(o)F(1)) was overexpressed in Escherichia coli and the intact c-subunit ring (TF(o)c-ring) was isolated by new procedures. One of the key improvement in this purification was the introduction of a His residue to each c-subunit that acts as a virtual His(10)-tag of the c-ring. After solubilization from membranes by sodium deoxycholate, the c-ring was purified by Ni-NTA affinity chromatography, followed by anion-exchange chromatography. The intactness of the isolated c-ring was confirmed by high-resolution clear native PAGE, sedimentation analysis, and H(+)-translocation activity. The isotope-labeled intact TF(o)c-ring was successfully purified in such an amount as enough for solid-state NMR measurements. The isolated TF(o)c-rings were reconstituted into lipid membranes. A solid-state NMR spectrum at a high quality was obtained with this membrane sample, revealing that this purification procedure was suitable for the investigation by solid-state NMR. The purification method developed here can also be used for other physicochemical investigations.     

Hip1-related mutant mice grow and develop normally but have accelerated spinal abnormalities and dwarfism in the absence of HIP1

In mice and humans, there are two known members of the Huntingtin interacting protein 1 (HIP1) family, HIP1 and HIP1-related (HIP1r). Based on structural and functional data, these proteins participate in the clathrin trafficking network. The inactivation of Hip1 in mice leads to spinal, hematopoietic, and testicular defects. To investigate the biological function of HIP1r, we generated a Hip1r mutant allele in mice. Hip1r homozygous mutant mice are viable and fertile without obvious morphological abnormalities. In addition, embryonic fibroblasts derived from these mice do not have gross abnormalities in survival, proliferation, or clathrin trafficking pathways. Altogether, this demonstrates that HIP1r is not necessary for normal development of the embryo or for normal adulthood and suggests that HIP1 or other functionally related members of the clathrin trafficking network can compensate for HIP1r absence. To test the latter, we generated mice deficient in both HIP1 and HIP1r. These mice have accelerated development of abnormalities seen in Hip1 -deficient mice, including kypholordosis and growth defects. The severity of the Hip1r/Hip1 double-knockout phenotype compared to the Hip1 knockout indicates that HIP1r partially compensates for HIP1 function in the absence of HIP1 expression, providing strong evidence that HIP1 and HIP1r have overlapping roles in vivo.     

Hierarchical organization of the plasma membrane: Investigations by single-molecule tracking vs. fluorescence correlation spectroscopy

Single-molecule tracking and fluorescence correlation spectroscopy (FCS) applied to the plasma membrane in living cells have allowed a number of unprecedented observations, thus fostering a new basic understanding of molecular diffusion, interaction, and signal transduction in the plasma membrane. It is becoming clear that the plasma membrane is a heterogeneous entity, containing diverse structures on nano-meso-scales (2-200 nm) with a variety of lifetimes, where certain membrane molecules stay together for limited durations. Molecular interactions occur in the time-dependent inhomogeneous two-dimensional liquid of the plasma membrane, which might be a key for plasma membrane functions.