[show abstract][hide abstract] ABSTRACT: The cellular prion protein (PrP(C)) was recently observed to co-purify with members of the LIV-1 subfamily of ZIP zinc transporters (LZTs), precipitating the surprising discovery that the prion gene family descended from an ancestral LZT gene. Here, we compared the subcellular distribution and biophysical characteristics of LZTs and their PrP-like ectodomains. When expressed in neuroblastoma cells, the ZIP5 member of the LZT subfamily was observed to be largely directed to the same subcellular locations as PrP(C) and both proteins were seen to be endocytosed through vesicles decorated with the Rab5 marker protein. When recombinantly expressed, the PrP-like domain of ZIP5 could be obtained with yields and levels of purity sufficient for structural analyses but it tended to aggregate, thereby precluding attempts to study its structure. These obstacles were overcome by moving to a mammalian cell expression system. The subsequent biophysical characterization of a homogeneous preparation of the ZIP5 PrP-like ectodomain shows that this protein acquires a dimeric, largely globular fold with an α-helical content similar to that of mammalian PrP(C). The use of a mammalian cell expression system also allowed for the expression and purification of stable preparations of Takifugu rubripes PrP-1, thereby overcoming a key hindrance to high-resolution work on a fish PrP(C).
PLoS ONE 01/2013; 8(9):e72446. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: Spinocerebellar ataxias (SCA) are a genetically heterogeneous group of neurodegenerative diseases characterised by progressive cerebellar ataxia, dysarthria and oculomotor abnormalities. Recently the prodynorphin (PDYN) gene was identified as the cause of SCA23 in four Dutch families displaying progressive gait and limb ataxia. In this study we aimed to assess the frequency of PDYN gene defects and extend the phenotype of SCA23 patients in a UK ataxia series and also in patients from Greece, Egypt and India. We sequenced the coding and flanking intronic regions of the PDYN gene in a total of 852 ataxia patients, of which 356 were sporadic with no family history, 320 had a positive family history, and 176 probands had a positive family history and at least one family member had also been investigated. We also analysed 190 patients with multiple-system atrophy with cerebellar features (MSA-C), a phenocopy of SCA23. We identified a novel putative pathogenic heterozygous missense variant in the PDYN gene in an early onset SCA patient with an unknown family history. This variant was not present in 570 matched British controls. This is the first study to screen for SCA23 in UK patients and confirms that PDYN mutations are a very rare cause of spinocerebellar ataxia, accounting for ~ 0.1 % of ataxia cases but perhaps with a higher frequency in pure cerebellar ataxia. Given the rarity of PDYN mutations, front-line diagnostic evaluation of UK familial and early onset pure spinocerebellar ataxia patients should focus on other known ataxia genes.
Journal of Neurology 10/2012; · 3.58 Impact Factor
[show abstract][hide abstract] ABSTRACT: The evolutionary origins of vertebrate prion genes had remained elusive until recently when multiple lines of evidence converged to the proposition that members of the prion gene family represent an ancient branch of a larger family of ZIP metal ion transporters. ( 1) A follow-up investigation which explored the mechanism of evolution in more detail led to the surprising conclusion that the emergence of the prion founder gene likely involved the reverse transcription of a spliced transcript of a LIV-1 ZIP predecessor gene. ( 2) The objective of this perspective is to discuss the possible significance of this reunion of ZIP and prion gene subfamilies for understanding the biology of the prion protein in health and disease. While a recent review article broadly introduced this area of research, ( 3) the emphasis here is to comment on some of the more pertinent concepts, experimental paradigms, ongoing developments and challenges.
[show abstract][hide abstract] ABSTRACT: We recently documented the co-purification of members of the LIV-1 subfamily of ZIP (Zrt-, Irt-like Protein) zinc transporters (LZTs) with the cellular prion protein (PrP(C)) and, subsequently, established that the prion gene family descended from an ancestral LZT gene. Here, we begin to address whether the study of LZTs can shed light on the biology of prion proteins in health and disease. Starting from an observation of an abnormal LZT immunoreactive band in prion-infected mice, subsequent cell biological analyses uncovered a surprisingly coordinated biology of ZIP10 (an LZT member) and prion proteins that involves alterations to N-glycosylation and endoproteolysis in response to manipulations to the extracellular divalent cation milieu. Starving cells of manganese or zinc, but not copper, causes shedding of the N1 fragment of PrP(C) and of the ectodomain of ZIP10. For ZIP10, this posttranslational biology is influenced by an interaction between its PrP-like ectodomain and a conserved metal coordination site within its C-terminal multi-spanning transmembrane domain. The transition metal starvation-induced cleavage of ZIP10 can be differentiated by an immature N-glycosylation signature from a constitutive cleavage targeting the same site. Data from this work provide a first glimpse into a hitherto neglected molecular biology that ties PrP to its LZT cousins and suggest that manganese or zinc starvation may contribute to the etiology of prion disease in mice.
Journal of Molecular Biology 06/2012; 422(4):556-74. · 3.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Hereditary spastic paraplegias (HSPs) are a clinically and genetically heterogeneous group of neurological diseases, which typically present with progressive lower extremity weakness and spasticity causing progressive walking difficulties. Complicating neurological or extraneurological features may be present.
We describe a 19-year-old male who was referred because of an action tremor of the hands; he later developed walking difficulties. Callosal atrophy was present on his cerebral magnetic resonance imaging scan, prompting genetic testing for SPG11, which revealed homozygous mutations.
The clinical features, differential diagnosis and management of SPG11, the most common form of autosomal recessive complicated HSP with a thin corpus callosum are discussed.
Tremor and other hyperkinetic movements (New York, N.Y.). 01/2012; 2.
[show abstract][hide abstract] ABSTRACT: To identify genes whose expressions in primary human trabecular meshwork (TM) cell cultures are affected by the transcription factor pituitary homeobox 2 (PITX2) and to identify genes that may have roles in glaucoma. Known glaucoma causing genes account for disease in a small fraction of patients, and we aimed at identification of other genes that may have subtle and accumulative effects not easily identifiable by a genetic approach.
Expression profiles derived using microarrays were compared between TM control cells and cells treated with PITX2 siRNAs using three protocols so as to minimize false positive and negative results. The first protocol was based on the commonly used B statistic. The second and third protocols were based on fold change in expression. The second protocol used a threshold of at least 2 fold change in expression, whereas the third protocol used ranking in fold change without setting a threshold. The likelihood of a selected gene being a true positive was considered to correlate with the number of protocols by which it was selected. By considering all genes that were selected by at least one protocol, the likelihood of false negatives was expected to decrease. Effects on a subset of selected genes were verified by real time PCR, western blots, and immunocytochemistry. Effects on ALDH1A1, were further pursued because its protein product, aldehyde dehydrogenase 1 family, member A1, has roles in oxidative stress and because oxidative stress is known to be relevant to the etiology of glaucoma.
The expression level of 41 genes was assessed by to be possibly affected by PITX2 knockdown. Twenty one genes were down-regulated and twenty were upregulated. The expression of five genes was assessed to be altered by all three analysis protocols. The five genes were DIRAS3 (DIRAS family, GTP-binding RAS-like 3), CXCL6 (chemokine (C-X-C motif) ligand 6), SAMD5 (sterile alpha motif domain containing 5), CBFB (core-binding factor, beta subunit), and MEIS2 (meis homeobox 2). Real time PCR experiments verified results on a subset of genes tested. Notably, the results were also confirmed in two independent TMs. Effects on CXCL6 and ALDH1A1 were also confirmed by western blots, and effects on ALDH1A1 were further shown by immunocytochemistry. Data consistent with PITX2 involvement in ALDH1A1 mediated response to oxidative stress were presented.
Bioinformatics tools revealed that the genes identified affect functions and pathways relevant to glaucoma. Involvement of PITX2 in expression of some of the genes and in some of the pathways is being reported here for the first time. As many of the genes identified have not been studied vis-à-vis glaucoma, we feel they introduce new candidates for understanding this devastating disease.