Robert Strome

Spatial learning in transgenic mice expressing human presenilin 1 (PS1) transgenes

Dominant mutations in the Presenilin 1 gene are linked to an aggressive, early-onset form of familial Alzheimers Disease (FAD). Spatial memory of transgenic (Tg) mice expressing either mutant (lines Tg(M146L)1, Tg(M146L)76, Tg(L286V)198) or wild type (line Tg(PS1wt)195) human PS1 transgenes was investigated in the Morris water maze (WM) test at 6 and 9 months of age. The results showed that the mutated Tg mice had increased swim speed when compared to non-Tg littermates or Tg PS1 wild type mice. The swim speed difference did not, however, significantly affect the spatial learning in the WM test and all groups showed comparable search paths during training and similar spatial bias during probe trials. When re-tested at 9 months, all mice showed significantly improved learning acquisition of spatial information. The lack of progressive spatial learning impairment in mice expressing the mutated human PS1 transgene in the WM does not preclude impairments in other cognitive tasks but suggests that full phenotypic expression of mutant PS1 alleles may require co-expression of human versions of other AD-associated genes.

LIV-1 ZIP Ectodomain Shedding in Prion-Infected Mice Resembles Cellular Response to Transition Metal Starvation

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.

A novel double mutation in FUS gene causing sporadic ALS.

It has been shown that mutations in the Fused in Sarcoma gene (FUS) could explain up to 5% of cases with familial amyotrophic lateral sclerosis (ALS). Our mutation analysis of FUS in a Canadian ALS patient of Chinese origin revealed an unusual novel heterozygous double point mutation (R514S/E516V) confirming that exon 15 is a mutation hot-spot. The substitutions are in cis position to each other and affect highly conserved codons in the RGG-rich region of the FUS protein. The absence of clinical signs of ALS in the relatives of the affected carrier could indicate that this mutation is incompletely penetrant or de novo. The pathologic significance of the R514S/E516V mutation was confirmed by immunohistochemistry. FUS-positive cytoplasmic inclusions were noted in a moderate number in neurons and abundantly in glial cells in the motor cortex and the brainstem. Of interest, a significant number of neuronal and glial FUS-positive inclusions were found in the tegmentum of the brainstem. Importantly, some neurons with inclusions showed retention of the normal nuclear FUS immunostaining.

Syrian hamster prion protein (PrPC) is expressed in photoreceptor cells of the adult retina

PrP(C), the cellular isoform of the prion protein (PrP) serves as a precursor to abnormal PrP isoforms which accumulate in diseases such as scrapie in sheep, and Creutzfeldt-Jakob disease in humans. Since prions can replicate in photoreceptors we surmised that PrP(C) must be expressed in these cells. Accordingly, monoclonal antisera directed against two epitopes of hamster PrP(C) produced retinal immunostaining in hamsters, and in mice bearing a hamster PrP transgene. Immunostaining was most prominent in the inner and outer segments of rod photoreceptors, coinciding with the earliest site of pathologic changes in scrapie-infected hamsters. These data define PrP(C) expression in an experimentally-accessible population of neurons and suggest that the retina may comprise a useful system for studying the biology of wild-type and mutant prion proteins.

A phosphorylation site in the Ftz homeodomain is required for activity

The Drosophila homeodomain‐containing protein Fushi tarazu (Ftz) is expressed sequentially in the embryo, first in alternate segments, then in specific neuroblasts and neurons in the central nervous system, and finally in parts of the gut. During these different developmental stages, the protein is heavily phosphorylated on different subsets of Ser and Thr residues. This stage‐specific phosphorylation suggests possible roles for signal transduction pathways in directing tissue‐specific Ftz activities. Here we show that one of the Ftz phosphorylation sites, T263 in the N‐terminus of the Ftz homeodomain, is phosphorylated in vitro by Drosophila embryo extracts and protein kinase A. In the embryo, mutagenesis of this site to the non‐phosphorylatable residue Ala resulted in loss of ftz‐dependent segments. Conversely, substitution of T263 with Asp, which is also non‐phosphorylatable, but which successfully mimics phosphorylated residues in a number of proteins, rescued the mutant phenotype. This suggests that T263 is in the phosphorylated state when functioning normally in vivo. We also demonstrate that the T263 substitutions of Ala and Asp do not affect Ftz DNA‐binding activity in vitro, nor do they affect stability or transcriptional activity in transfected S2 cells. This suggests that T263 phosphorylation is most likely required for a homeodomain‐mediated interaction with an embryonically expressed protein.

The prion protein modulates A-type K+ currents mediated by Kv4.2 complexes through Dipeptidyl Aminopeptidase-like protein 6

Background: Prion protein (PrP) interacts with dipeptidyl aminopeptidase-like protein 6 (DPP6), but the functional significance was unknown. Results: PrP formed complexes with and impacted the function of potassium channels containing DPP6 and Kv4.2. Conclusion: PrP modulates voltage-dependent and kinetic properties of Kv4.2 channels. Significance: This could explain a phenotype of PrP knock-out mice and the effects of amyloid β oligomers. Widely expressed in the adult central nervous system, the cellular prion protein (PrPC) is implicated in a variety of processes, including neuronal excitability. Dipeptidyl aminopeptidase-like protein 6 (DPP6) was first identified as a PrPC interactor using in vivo formaldehyde cross-linking of wild type (WT) mouse brain. This finding was confirmed in three cell lines and, because DPP6 directs the functional assembly of K+ channels, we assessed the impact of WT and mutant PrPC upon Kv4.2-based cell surface macromolecular complexes. Whereas a Gerstmann-Sträussler-Scheinker disease version of PrP with eight extra octarepeats was a loss of function both for complex formation and for modulation of Kv4.2 channels, WT PrPC, in a DPP6-dependent manner, modulated Kv4.2 channel properties, causing an increase in peak amplitude, a rightward shift of the voltage-dependent steady-state inactivation curve, a slower inactivation, and a faster recovery from steady-state inactivation. Thus, the net impact of wt PrPC was one of enhancement, which plays a critical role in the down-regulation of neuronal membrane excitability and is associated with a decreased susceptibility to seizures. Insofar as previous work has established a requirement for WT PrPC in the Aβ-dependent modulation of excitability in cholinergic basal forebrain neurons, our findings implicate PrPC regulation of Kv4.2 channels as a mechanism contributing to the effects of oligomeric Aβ upon neuronal excitability and viability.

Presenilin Proteins and the Pathogenesis of Early-Onset Familial Alzheimer’s Disease: β-Amyloid Production and Parallels to Prion Diseases

In contrast to rare mutations in the amyloid presursor protein (APP) gene, missense mutations in the presenilin 1 (PS1) and presenilin 2 (PS2) genes, on chromosomes 14 and 1 respectively, are the most common causes of early-onset familial Alzheimer’s disease (AD)(Sherrington et al. 1995)(Rogaev et al. 1995)(Levy-Lahad et al. 1995). Presenilin genes encode proteins with at least seven putative transmembrane domains and an extruded cytoplasmic “loop”, the latter with a preponderance of acidic amino acid residues: these proteins are expressed in a variety of cell types. While the physiologic function of these genes is unknown, their protein products have been demonstrated to accumulate in intracellular sites including the endoplasmic reticulum, and the Golgi apparatus. Similarly, the mechanism by which the 30 different point mutations have been identified in PS1 and PS2 to date cause the clinical and neuropathological hallmarks of Alzheimer disease is unknown. However, fibroblasts from heterozygous carriers of PS1 and PS2 mutations secrete increased levels of the amyloidogenic long-tailed amyloid β-peptides ending at residues 42 or 43 (Aβ42)(Martin 1995)(Scheuner et al. 1996). Increased levels of Aβ42 and other Aβ-peptides can also be measured in postmortem brain tissue from human patients dying with early-onset FAD associated with PS1 mutations(Lemere et al. 1996). To determine whether overproduction of Aβ peptides occurs in brain as an early biochemical event prior to the onset of neurodegeneration, we constructed transgenic mice with either mutant or wild-type human PS1 and mated them with another line of transgenic mice overexpressing wild-type human βAPP695 under the control of the same transcriptional regulatory element. These studies reveal that mutant PS1 transgenes but not wild-type PS1 transgenes act in a dominant fashion to programme over-production of long-tailed Aβ42 peptides in brain, and that this biochemical difference is present by at least 2–4 months of age and in the absence of any detectable neuropathologic lesions. These advances in our understanding of presenilin function are discussed in relation to the two schools of thought on AD pathogenesis, “tau-ist”and “saptist”, and also with regards the hypothesis that similarities between AD and prion diseases reflect the existence of shared pathogenic pathways.

P1-079: Human gamma-secretase reconstituted in yeast via co-expression of presenilin, nicastrin, APH1A and PEN2 deviates from the prototype and implies the existence of auxiliary components

loid levels by ELISA. Results: Analysis of AChE activity in hippocampus of lesioned mice revealed significant decreases by 68% in wild-type (WT) and by 88% in transgenic mice. In the cortex, only transgenic lesioned mice showed significant reductions in AChE activity (39%). Transgenic mice, regardless of the lesion, showed important spatial memory impairments in the Morris water maze, while lesioned WT mice showed partial learning deficits. The novel object recognition test revealed in lesioned mice, both WT and transgenic, significant cognitive deficits. -amyloid (A )1-40 and A 1-42 levels were found to be increased with age in transgenic mice. These increases were significantly higher in cholinergically lesioned transgenic animals (soluble A 1-40 by 60%; fibrillar A 1-40 by 73%; soluble A 1-42 by 14-fold; fibrillar A 1-42 by 2.3-fold). Levels of A 1-40 (soluble and fibrillar) were correlated to retention time in the Morris water maze in transgenic mice, but this correlation was lost in the lesioned group. Conclusions: Cholinergic neurons of Tg2576 mice appear to be especially vulnerable to a cholinergic lesion. Basal forebrain cholinergic dysfunction favours the amyloidogenic route of APP processing in Tg2576 mice by increasing A species, which does not lead to further cognitive impairments.