Eric Karran

A mutation in Alzheimer's disease destroying a splice acceptor site in the presenilin-1 gene

A series of mutations has been reported in the presenilin-1 (PS-1) gene which cause early onset Alzheimers disease (AD). The mutations reported to date have encoded missense mutations which alter residues conserved between PS-1 and the presenilin-2 (PS-2) gene. We have recently determined the intron/exon structure of the PS-1 gene and this information has been used to identify a mutation in the splice acceptor site for exon 9 in a family with early onset AD. Amplification of cDNA from lymphoblasts of affected individuals revealed that the effect of the mutation was to cause splicing out of exon 9, however it does not change the open reading frame of the mRNA. The importance of this observation is discussed.

Complete analysis of the presenilin 1 gene in early onset Alzheimer's disease

The presenilin 1 gene has recently been identified as the locus on chromosome 14 which is responsible for a large proportion of early onset, autosomal dominantly inherited Alzheimers disease (AD). We have elucidated the intron/exon structure of the gene and designed intronic primers to enable direct sequencing of the entire coding region (10 exons) of the presenilin gene in a large number of families. This strategy has enabled us to find a further two novel mutations in the gene. We discuss the distribution of mutations and the proportions of autosomal dominant AD with a mean age of onset below 60 years caused by mutations in this gene.

ALZHEIMER'S DISEASE-ASSOCIATED PRESENILIN 1 IN NEURONAL CELLS : EVIDENCE FOR LOCALIZATION TO THE ENDOPLASMIC RETICULUM-GOLGI INTERMEDIATE COMPARTMENT

The recently identified Alzheimers disease‐associated presenilin 1 and 2 (PS1 and PS2) genes encode two homologous multi membrane‐spanning proteins. Rabbit antibodies to the N‐terminal domain of PS1 detected PS1 in human neuroblastoma SH‐SY5Y wild type and PS1 transfectants (SY5Y‐PS1) as well as in mouse P19, in CHO‐K1 and CHO‐APP770 transfected cells, in rat cerebellar granule and hippocampal neurons, and astrocytes. Immunoblotting detected full‐length protein of 50 kDa, and a major presumptive cleavage product of 30 kDa. The immunofluorescence pattern resembled labeling of the endoplasmic reticulum‐Golgi intermediate compartment (ERGIC) marker protein ERGIC‐53. PS1 distribution showed slight condensation after brefeldin A and more marked condensation after incubation of cells at 16°C, characteristic of the ERGIC compartment. Double labeling showed colocalization of ERGIC‐53 with PS1 in the SY5Y‐PS1 cells. PS1 labeling of SY5Y‐PS1 and P19 cells showed overlap of the cis‐Golgi marker p210 and colocalization with p210 after brefeldin A which causes redistribution of p210 to the ERGIC. Expression of PS1 did not change in level or cellular distribution during development of neurons in culture. Double labeling for the amyloid precursor protein (APP) and PS1 on SY5Y‐PS1 cells and CHO‐APP770 cells showed some overlap under control conditions. These results indicate that PS1 is a resident protein of the ERGIC and could be involved in trafficking of proteins, including APP, between the ER and Golgi compartments. J. Neurosci. Res. 49:719–731, 1997.

Proteolytic Fragments of Alzheimer’s Disease‐Associated Presenilin 1 Are Present in Synaptic Organelles and Growth Cone Membranes of Rat Brain

Abstract: Previous studies have demonstrated the molecular linkage of three causative genes for early‐onset Alzheimer’s disease: the presenilin 1 gene on chromosome 14, the presenilin 2 gene on chromosome 1, and the amyloid precursor protein gene on chromosome 21. In the present study, we have investigated the distributions of the ∼20‐kDa C‐terminal and ∼30‐kDa N‐terminal fragments of presenilin 1 and the amyloid precursor protein in rat brain and compared them with the distribution of several marker proteins. The fragments of presenilin 1 are present in synaptic plasma membranes, neurite growth cone membranes, and small synaptic vesicles of rat brain. Both proteolytic fragments are coenriched in the corresponding tissue fractions. Based on this observation, it seems likely that N‐ and C‐terminal presenilin 1 fragments form a functional unit while remaining associated. In contrast to a predominant subcellular localization of presenilin 1 to the endoplasmic reticulum and Golgi apparatus in different cell lines, our results indicate that rat brain presenilin 1 fragments exit from these biosynthetic compartments to reach synaptic organelles in neurons.

Structure and alternative splicing of the presenilin-2 gene.

Missense mutations in the presenilin-1 (PS-1) and presenilin-2 (PS-2) genes have been shown to be causes of autosomal dominant Alzheimers disease (the AD3 and AD4 loci, respectively). Alternative splicing has previously been reported in the PS-1 gene. In this study, elucidation of intron/exon boundary sequences revealed that PS-2 is encoded by 10 coding exons. In addition, PS-2 cDNA cloning and RT-PCR using RNA from a variety of normal tissues revealed the presence of alternatively spliced products. These products included species with in frame omissions of exon 8 and simultaneous omissions of exons 3 and 4.

Alzheimer's disease : Correlation of the suppression of β-amyloid Peptide secretion from cultured cells with inhibition of the chymotrypsin-like activity of the proteasome

Abstract: Peptide aldehyde inhibitors of the chymotrypsin‐like activity of the proteasome (CLIP) such as N‐acetyl‐Leu‐Leu‐Nle‐H (or ALLN) have been shown previously to inhibit the secretion of β‐amyloid peptide (Aβ) from cells. To evaluate more fully the role of the proteasome in this process, we have tested the effects on Aβ formation of a much wider range of peptide‐based inhibitors of CLIP than published previously. The inhibitors tested included several peptide boronates, some of which proved to be the most potent peptide‐based inhibitors of β‐amyloid production reported so far. We found that the ability of the peptide aldehyde and boronate inhibitors to suppress Aβ formation from cells correlated extremely well with their potency as CLIP inhibitors. Thus, we conclude that the proteasome may be involved either directly or indirectly in Aβ formation.

An unbiased approach to identifying tau kinases that phosphorylate tau at sites associated with Alzheimer disease.

Background: Abnormally hyperphosphorylated tau is present in neurofibrillary tangles in Alzheimer disease. Results: Key kinases that phosphorylate tau at Alzheimer disease-specific epitopes have been identified in a cell-based screen of kinases. Conclusion: GSK3α, GSK3β, and MAPK13 were the most active tau kinases. Significance: Findings identify novel tau kinases and novel pathways that may be relevant for Alzheimer disease and other tauopathies. Neurofibrillary tangles, one of the hallmarks of Alzheimer disease (AD), are composed of paired helical filaments of abnormally hyperphosphorylated tau. The accumulation of these proteinaceous aggregates in AD correlates with synaptic loss and severity of dementia. Identifying the kinases involved in the pathological phosphorylation of tau may identify novel targets for AD. We used an unbiased approach to study the effect of 352 human kinases on their ability to phosphorylate tau at epitopes associated with AD. The kinases were overexpressed together with the longest form of human tau in human neuroblastoma cells. Levels of total and phosphorylated tau (epitopes Ser(P)-202, Thr(P)-231, Ser(P)-235, and Ser(P)-396/404) were measured in cell lysates using AlphaScreen assays. GSK3α, GSK3β, and MAPK13 were found to be the most active tau kinases, phosphorylating tau at all four epitopes. We further dissected the effects of GSK3α and GSK3β using pharmacological and genetic tools in hTau primary cortical neurons. Pathway analysis of the kinases identified in the screen suggested mechanisms for regulation of total tau levels and tau phosphorylation; for example, kinases that affect total tau levels do so by inhibition or activation of translation. A network fishing approach with the kinase hits identified other key molecules putatively involved in tau phosphorylation pathways, including the G-protein signaling through the Ras family of GTPases (MAPK family) pathway. The findings identify novel tau kinases and novel pathways that may be relevant for AD and other tauopathies.

NOVEL NLRP3 INFLAMMASOME INHIBITORS: EVALUATION IN IL-1β RELEASE AND HIGH-CONTENT ASC-SPECK CELLULAR ASSAYS

Background: Inflammation has been correlated to Alzheimer’s disease (AD) and a better understanding of its mechanisms may help to develop new therapies for AD. We have recently demonstrated that neutrophils infiltrate the brain and produce neutrophil extracellular traps (NETs) in subjects with AD and mice with AD-like disease. Furthermore, inhibition of neutrophil trafficking has therapeutic effect in animal models of AD. Recent studies have shown that protein arginine deiminases (PADs) mediate NET formation and have a role in innate immunity responses. PAD inhibitors have demonstrated remarkable efficacy in numerous inflammatory diseases including rheumatoid arthritis, lupus, ulcerative colitis, multiple sclerosis and atherosclerosis. The aim of our study was to test the potential therapeutic effect of PAD inhibition in AD models. Methods:BB-Cl-amidine, a panPAD inhibitor, and GSK199, a PAD4 specific inhibitor were used to study the role of PADs in vitro and in vivo. The role of PADs in rapid adhesion in vitro was assessed in adhesion assays on integrin ligands. Epifluorescence intravital microscopy and two-photon microscopy were performed to study the role of PADs in neutrophil adhesion in vivo. 5xFAD mice, which develop amyloid pathology, and 3xTg-AD mice, which develop both amyloid and tau pathology, were used to test the effect of PAD inhibition on disease. Results: Treatment of neutrophils with PAD inhibitors reduced integrindependent rapid adhesion triggered by chemoattractants and TNF. PAD inhibition also reduced rolling interactions and drastically inhibited firm adhesion in inflamed brain microcirculation. Moreover, two-photonmicroscopy performed in the brain of ADmice showed a drastic reduction of adhesion and intravascular crawling of neutrophils treated with BB-Cl-amidine compared to untreated cells, suggesting that PADs control neutrophil trafficking in the AD brain. Notably, treatment with BB-Cl-amidine of 3xTg-AD and 5xFAD mice led to a significant restoration of memory in Y maze and contextual fear conditioning behavioral tests when compared to control animals. Neuropathological studies showed that treatment with BB-Cl-amidine reduced microglial activation and amyloid deposition compared to control animals. Conclusions:PAD inhibition interferes with neutrophil trafficking and ameliorates cognitive impairment and neuropathological changes in animal models of AD, suggesting PADs represent new therapeutic targets in AD.

Current academic drug discovery initiatives

evaluated CSF samples from subsets of two additional unique cohorts of HC and AD, including samples from the Australian Imaging Biomarkers and Lifestyle Study of Aging (AIBL) and the Oxford Project to Investigate Memory and Ageing (OPTIMA) studies (J Alzheimers Dis. 44:525-539). Conclusions:These cohorts provide critical genotype, biochemical, imaging, cognitive and functional data with which to assess oligomers as potential surrogates of disease state. The ability of oligomers to differentiate between HC and AD, and the assessment of their performance in comparison with amyloid and tau, will be reported.

Report from the Alzheimer’s Research UK Conference 2015

On 10–11 March 2015 University College London hosted the annual Alzheimer’s Research UK Conference. This report provides an overview of the presentations and discussions that took place.