R. Johnsen

Dystrophin expression in the mdx mouse after localised and systemic administration of a morpholino antisense oligonucleotide.

Duchenne and Becker muscular dystrophies are allelic disorders arising from mutations in the dystrophin gene. Duchenne muscular dystrophy is characterised by an absence of functional protein, while Becker muscular dystrophy is usually caused by in‐frame deletions allowing synthesis of some functional protein. Treatment options are limited, and we are investigating the potential of transcript manipulation to overcome disease‐causing mutations. Antisense oligonucleotides have been used to induce specific exon removal during processing of the dystrophin primary transcript and thereby by‐pass protein‐truncating mutations. The antisense oligonucleotide chemistry most widely used to alter pre‐mRNA processing is 2′‐O‐methyl‐modified bases on a phosphorothioate backbone.

Prevalence of amyloid-β deposition in the cerebral cortex in Parkinson's disease

The pathological basis for the dementia which occurs in 20 to 40% of patients with idiopathic Parkinsons disease (PD) remains uncertain. In the present postmortem study, we compared the prevalence and severity of parenchymal and vascular amyloid‐β (Aβ) deposition in the cerebral cortex in a group of 57 PD brains, including 13 cases with dementia, and in 100 control brains. A higher proportion of PD brains had vascular Aβ deposition, whereas the proportions and severity of parenchymal Aβ were similar in the PD and control groups. There was a poor correlation between Aβ deposition and neurofibrillary tangles which were present in only small numbers in a minority of cases. Cortical Aβ deposition was present in only 6 of the 13 cases with dementia and only 3 fulfilled the Consortium to Establish a Registry for Alzheimers Disease (CERAD) criteria for definite Alzheimers disease. The present findings confirm that dementia in PD is only infrequently due to fully established Alzheimers disease. However, vascular and parenchymal Aβ deposition could still contribute to dementia and cognitive decline when combined with other changes such as α‐synuclein deposition in the cerebral cortex and cortical Lewy bodies.

Prevalence of Stroke in Parkinson's Disease: A Postmortem Study

The results of previous epidemiological studies of the relationship between Parkinsons disease and stroke have been conflicting; some showing a reduced risk of ischaemic and haemorrhagic stroke during life, and others indicating an increased likelihood of stroke‐related death. We compared the frequency of cerebral infarcts and haemorrhages at postmortem in 100 cases of pathologically verified idiopathic Parkinsons disease and 100 age‐matched control brains. No significant differences were found in the numbers of infarcts or haemorrhages or stroke‐related deaths between the two groups. Our findings do not indicate either a protective effect against stroke, or a greater susceptibility to death from stroke, in the population studied.

Differential effects of dietary fatty acids on the cerebral distribution of plasma-derived apo B lipoproteins with amyloid-β

Some dietary fats are a risk factor for Alzheimers disease (AD) but the mechanisms for this association are presently unknown. In the present study we showed in wild-type mice that chronic ingestion of SFA results in blood-brain barrier (BBB) dysfunction and significant delivery into the brain of plasma proteins, including apo B lipoproteins that are endogenously enriched in amyloid-beta (Abeta). Conversely, the plasma concentration of S100B was used as a marker of brain-to-blood leakage and was found to be increased two-fold because of SFA feeding. Consistent with a deterioration in BBB integrity in SFA-fed mice was a diminished cerebrovascular expression of occludin, an endothelial tight junction protein. In contrast to SFA-fed mice, chronic ingestion of MUFA or PUFA had no detrimental effect on BBB integrity. Utilising highly sensitive three-dimensional immunomicroscopy, we also showed that the cerebral distribution and co-localisation of Abeta with apo B lipoproteins in SFA-fed mice are similar to those found in amyloid precursor protein/presenilin-1 (APP/PS1) amyloid transgenic mice, an established murine model of AD. Moreover, there was a strong positive association of plasma-derived apo B lipoproteins with cerebral Abeta deposits. Collectively, the findings of the present study provide a plausible explanation of how dietary fats may influence AD risk. Ingestion of SFA could enhance peripheral delivery to the brain of circulating lipoprotein-Abeta and exacerbate the amyloidogenic cascade.

Expanding the phenotype of GMPPB mutations

Dystroglycanopathies are a heterogeneous group of diseases with a broad phenotypic spectrum ranging from severe disorders with congenital muscle weakness, eye and brain structural abnormalities and intellectual delay to adult-onset limb-girdle muscular dystrophies without mental retardation. Most frequently the disease onset is congenital or during childhood. The exception is FKRP mutations, in which adult onset is a common presentation. Here we report eight patients from five non-consanguineous families where next generation sequencing identified mutations in the GMPPB gene. Six patients presented as an adult or adolescent-onset limb-girdle muscular dystrophy, one presented with isolated episodes of rhabdomyolysis, and one as a congenital muscular dystrophy. This report expands the phenotypic spectrum of GMPPB mutations to include limb-girdle muscular dystrophies with adult onset with or without intellectual disability, or isolated rhabdomyolysis.

Three-dimensional colocalization analysis of plasma-derived apolipoprotein B with amyloid plaques in APP/PS1 transgenic mice.

Parenchymal accumulation of amyloid-beta (Aβ) is a hallmark pathological feature of Alzheimer’s disease. An emerging hypothesis is that blood-to-brain delivery of Aβ may increase with compromised blood–brain barrier integrity. In plasma, substantial Aβ is associated with triglyceride-rich lipoproteins (TRLs) secreted by the liver and intestine. Utilizing apolipoprotein B as an exclusive marker of hepatic and intestinal TRLs, here we show utilizing an highly sensitive 3-dimensional immuno-microscopy imaging technique, that in APP/PS1 amyloid transgenic mice, concomitant with substantially increased plasma Aβ, there is a significant colocalization of apolipoprotein B with cerebral amyloid plaque. The findings are consistent with the possibility that circulating lipoprotein-Aβ contributes to cerebral amyloidosis.

Targeted Exon Skipping to Address “Leaky” Mutations in the Dystrophin Gene

Protein-truncating mutations in the dystrophin gene lead to the progressive muscle wasting disorder Duchenne muscular dystrophy, whereas in-frame deletions typically manifest as the milder allelic condition, Becker muscular dystrophy. Antisense oligomer-induced exon skipping can modify dystrophin gene expression so that a disease-associated dystrophin pre-mRNA is processed into a Becker muscular dystrophy-like mature transcript. Despite genomic deletions that may encompass hundreds of kilobases of the gene, some dystrophin mutations appear “leaky”, and low levels of high molecular weight, and presumably semi-functional, dystrophin are produced. A likely causative mechanism is endogenous exon skipping, and Duchenne individuals with higher baseline levels of dystrophin may respond more efficiently to the administration of splice-switching antisense oligomers. We optimized excision of exons 8 and 9 in normal human myoblasts, and evaluated several oligomers in cells from eight Duchenne muscular dystrophy patients with deletions in a known “leaky” region of the dystrophin gene. Inter-patient variation in response to antisense oligomer induced skipping in vitro appeared minimal. We describe oligomers targeting exon 8, that unequivocally increase dystrophin above baseline in vitro, and propose that patients with leaky mutations are ideally suited for participation in antisense oligomer mediated splice-switching clinical studies.

Prevalence of cerebral vascular amyloid-β deposition and stroke in an aging Australian population : a postmortem study

Cerebral amyloid angiopathy (CAA) is a putative risk factor for lobar cerebral haemorrhage and infarction in the elderly. However, the prevalence of stroke in a population with CAA is not known. Amyloid-beta immunohistochemistry was used to assess CAA prevalence as a function of age, and the relationship between CAA and stroke in 100 individuals aged 50-91 years who died unexpectedly and had a Coroners postmortem. Blocks were taken from several cortical areas and from areas of infarction or haemorrhage. Parenchymal Abeta was first found in the 6th decade, whereas vascular Abeta did not appear until the 7th decade. The prevalence of both vascular and parenchymal Abeta increased with age to a maximum in the 9th decade. The age at onset of vascular Abeta deposition was similar to that in an English study of CAA but a decade later than in Japanese studies. There was no association between the presence of vascular Abeta and cerebral haemorrhage or infarction. The findings indicate differences in the time-course of vascular and parenchymal Abeta deposition with age, as well as racial differences. The lack of association between vascular Abeta and cerebral haemorrhage or infarction indicates that, in the present population, CAA was usually asymptomatic.

Dystrophin Isoform Induction In Vivo by Antisense-mediated Alternative Splicing

Antisense oligomer-induced manipulation of dystrophin pre-mRNA processing can remove exons carrying mutations, or exclude exons flanking frameshifting mutations, and restore dystrophin expression in dystrophinopathy models and in Duchenne muscular dystrophy (DMD) patients. Splice intervention can also be used to manipulate the normal dystrophin pre-mRNA processing and ablate dystrophin expression in wild-type mice, with signs of pathology being induced in selected muscles within 4 weeks of commencing treatment. The disruption of normal dystrophin pre-mRNA processing to alter the reading frame can be very efficient and offers an alternative mechanism to RNA silencing for gene suppression. In addition, it is possible to remove in-frame exon blocks from the DMD gene transcript and induce specific dystrophin isoforms that retain partial functionality, without having to generate transgenic animal models. Specific exon removal to yield in-frame dystrophin transcripts will facilitate mapping of functional protein domains, based upon exon boundaries, and will be particularly relevant where there is either limited, or conflicting information as to the consequences of in-frame dystrophin exon deletions on the clinical severity and progression of the dystrophinopathy.

Synergistic effects of high fat feeding and apolipoprotein E deletion on enterocytic amyloid-beta abundance.

BackgroundAmyloid-β (Aβ), a key protein found in amyloid plaques of subjects with Alzheimers disease is expressed in the absorptive epithelial cells of the small intestine. Ingestion of saturated fat significantly enhances enterocytic Aβ abundance whereas fasting abolishes expression. Apolipoprotein (apo) E has been shown to directly modulate Aβ biogenesis in liver and neuronal cells but its effect in enterocytes is not known. In addition, apo E modulates villi length, which may indirectly modulate Aβ as a consequence of differences in lipid absorption. This study compared Aβ abundance and villi length in wild-type (WT) and apo E knockout (KO) mice maintained on either a low-fat or high-fat diet. Wild-type C57BL/6J and apo E KO mice were randomised for six-months to a diet containing either 4% (w/w) unsaturated fats, or chow comprising 16% saturated fats and 1% cholesterol. Quantitative immunohistochemistry was used to assess Aβ abundance in small intestinal enterocytes. Apo E KO mice given the low-fat diet had similar enterocytic Aβ abundance compared to WT controls.ResultsThe saturated fat diet substantially increased enterocytic Aβ in WT and in apo E KO mice, however the effect was greater in the latter. Villi height was significantly greater in apo E KO mice than for WT controls when given the low-fat diet. However, WT mice had comparable villi length to apo E KO when fed the saturated fat and cholesterol enriched diet. There was no effect of the high-fat diet on villi length in apo E KO mice.ConclusionThe findings of this study are consistent with the notion that lipid substrate availability modulates enterocytic Aβ. Apo E may influence enterocytic lipid availability by modulating absorptive capacity.