Pathology Associated with AAV Mediated Expression of Beta Amyloid or C100 in Adult Mouse Hippocampus and Cerebellum

Accumulation of beta amyloid (Ab) in the brain is a primary feature of Alzheimer’s disease (AD) but the exact molecular mechanisms by which Ab exerts its toxic actions are not yet entirely clear. We documented pathological changes 3 and 6 months after localised injection of recombinant, bi-cistronic adeno-associated viral vectors (rAAV2) expressing human Ab40- GFP, Ab42-GFP, C100-GFP or C100V717F-GFP into the hippocampus and cerebellum of 8 week old male mice. Injection of all rAAV2 vectors resulted in wide-spread transduction within the hippocampus and cerebellum, as shown by expression of transgene mRNA and GFP protein. Despite the lack of accumulation of Ab protein after injection with AAV vectors, injection of rAAV2-Ab42-GFP and rAAV2- C100V717F-GFP into the hippocampus resulted in significantly increased microgliosis and altered permeability of the blood brain barrier, the latter revealed by high levels of immunoglobulin G (IgG) around the injection site and the presence of IgG positive cells. In comparison, injection of rAAV2-Ab40-GFP and rAAV2-C100-GFP into the hippocampus resulted in substantially less neuropathology. Injection of rAAV2 vectors into the cerebellum resulted in similar types of pathological changes, but to a lesser degree. The use of viral vectors to express different types of Ab and C100 is a powerful technique with which to examine the direct in vivo consequences of Ab expression in different regions of the mature nervous system and will allow experimentation and analysis of pathological AD-like changes in a broader range of species other than mouse

The Guinea Pig as a Model for Sporadic Alzheimer\u27s Disease (AD): The Impact of Cholesterol Intake on Expression of AD-Related Genes

We investigated the guinea pig, Cavia porcellus, as a model for Alzheimer’s disease (AD), both in terms of the conservation of genes involved in AD and the regulatory responses of these to a known AD risk factor - high cholesterol intake. Unlike rats and mice, guinea pigs possess an Ab peptide sequence identical to human Ab. Consistent with the commonality between cardiovascular and AD risk factors in humans, we saw that a high cholesterol diet leads to up-regulation of BACE1 (b-secretase) transcription and down-regulation of ADAM10 (a-secretase) transcription which should increase release of Ab from APP. Significantly, guinea pigs possess isoforms of AD-related genes found in humans but not present in mice or rats. For example, we discovered that the truncated PS2V isoform of human PSEN2, that is found at raised levels in AD brains and that increases c-secretase activity and Ab synthesis, is not uniquely human or aberrant as previously believed. We show that PS2V formation is up-regulated by hypoxia and a high-cholesterol diet while, consistent with observations in humans, Ab concentrations are raised in some brain regions but not others. Also like humans, but unlike mice, the guinea pig gene encoding tau, MAPT, encodes isoforms with both three and four microtubule binding domains, and cholesterol alters the ratio of these isoforms. We conclude that AD-related genes are highly conserved and more similar to human than the rat or mouse. Guinea pigs represent a superior rodent model for analysis of the impact of dietary factors such as cholesterol on the regulation of AD-related genes

Validation and characterisation of a novel peptide that binds monomeric and aggregated beta-amyloid and inhibits the formation of neurotoxic oligomers

Although the formation of β-amyloid (Aβ) deposits in the brain is a hallmark of Alzheimer disease (AD), the soluble oligomers rather than the mature amyloid fibrils most likely contribute to Aβ toxicity and neurodegeneration. Thus, the discovery of agents targeting soluble Aβ oligomers is highly desirable for early diagnosis prior to the manifestation of a clinical AD phenotype and also more effective therapies. We have previously reported that a novel 15-amino acid peptide (15-mer), isolated via phage display screening, targeted Aβ and attenuated its neurotoxicity (Taddei, K., Laws, S. M., Verdile, G., Munns, S., D'Costa, K., Harvey, A. R., Martins, I. J., Hill, F., Levy, E., Shaw, J. E., and Martins, R. N. (2010) Neurobiol. Aging 31, 203–214). The aim of the current study was to generate and biochemically characterize analogues of this peptide with improved stability and therapeutic potential. We demonstrated that a stable analogue of the 15-amino acid peptide (15M S.A.) retained the activity and potency of the parent peptide and demonstrated improved proteolytic resistance in vitro (stable to t = 300 min, c.f. t = 30 min for the parent peptide). This candidate reduced the formation of soluble Aβ42 oligomers, with the concurrent generation of non-toxic, insoluble aggregates measuring up to 25–30 nm diameter as determined by atomic force microscopy. The 15M S.A. candidate directly interacted with oligomeric Aβ42, as shown by coimmunoprecipitation and surface plasmon resonance/Biacore analysis, with an affinity in the low micromolar range. Furthermore, this peptide bound fibrillar Aβ42 and also stained plaques ex vivo in brain tissue from AD model mice. Given its multifaceted ability to target monomeric and aggregated Aβ42 species, this candidate holds promise for novel preclinical AD imaging and therapeutic strategies

The Guinea Pig as a model for sporadic Alzheimer's Disease (AD): the impact of cholesterol intake on expression of AD-related genes

Extent: 12p.We investigated the guinea pig, Cavia porcellus, as a model for Alzheimer’s disease (AD), both in terms of the conservation of genes involved in AD and the regulatory responses of these to a known AD risk factor - high cholesterol intake. Unlike rats and mice, guinea pigs possess an Aβ peptide sequence identical to human Aβ. Consistent with the commonality between cardiovascular and AD risk factors in humans, we saw that a high cholesterol diet leads to up-regulation of BACE1 (β-secretase) transcription and down-regulation of ADAM10 (α-secretase) transcription which should increase release of Aβ from APP. Significantly, guinea pigs possess isoforms of AD-related genes found in humans but not present in mice or rats. For example, we discovered that the truncated PS2V isoform of human PSEN2, that is found at raised levels in AD brains and that increases γ-secretase activity and Aβ synthesis, is not uniquely human or aberrant as previously believed. We show that PS2V formation is up-regulated by hypoxia and a high-cholesterol diet while, consistent with observations in humans, Aβ concentrations are raised in some brain regions but not others. Also like humans, but unlike mice, the guinea pig gene encoding tau, MAPT, encodes isoforms with both three and four microtubule binding domains, and cholesterol alters the ratio of these isoforms. We conclude that AD-related genes are highly conserved and more similar to human than the rat or mouse. Guinea pigs represent a superior rodent model for analysis of the impact of dietary factors such as cholesterol on the regulation of AD-related genes.Mathew J. Sharman, Seyyed H. Moussavi Nik, Mengqi M. Chen, Daniel Ong, Linda Wijaya, Simon M. Laws, Kevin Taddei, Morgan Newman, Michael Lardelli, Ralph N. Martins, Giuseppe Verdil

Diabetes insipidus in neuropsychiatric-systemic lupus erythematosus patient

Systemic lupus erythematosus (SLE) is an idiopathic autoimmune chronic inflammatorydisease that is unique in its diversity of clinical manifestations, variability of disease’s progression, and prognosis. The disease is characterized by the remission and multiple flare-ups in between the chronic phase that may affect many organ systems.The prevalence of SLE in the US population is 1:1000 with a woman to man ratio of about 9-14:1. At Cipto Mangunkusumo Hospital, Jakarta in 2002, there was 1.4% cases of SLE of the total number of patients at the Rheumatology Clinic. Neuropsychiatric manifestations of SLE (NP-SLE) have a high mortality and morbidity rates. The incidence of NP-SLE ranges 18-61%. Diagnosis of NP-SLE is difficult because there is no specific laboratory examination. Accordingly, in all SLE patients with central nervous system (CNS) dysfunction, additional tests will be necessary to confirm an NP-SLE diagnosis and exclude other causes. Similar to diabetes insipidus, SLE is a systemic disease which affects many organ systems, one being the endocrine system. No data has specified the occurrence rate of diabetes insipidus in SLE patients. This disease arises from a number of factors able to interfere with the mechanism of neurohypophyseal renal reflex resulting in the body’s failure to convert water.3 There are three general forms of the disease, a polydipsicpolyuric syndrome caused by partial/complete vasopressin deficiency (central-diabetes-insipidus/CDI), vasopressin resistance of the kidney tubules (nephrogenic-diabetes-insipidus/NDI), and primary polydipsia. CDI occurs in about 1 in 25,000 person

Transduction in the hippocampus and cerebellum as indicated by GFP expression.

<p>Low magnification images show widespread GFP protein throughout the hippocampus (<b>A</b>) and cerebellar cortex (<b>C</b>). GFP protein was also observed in the contralateral hippocampus (<b>B</b>). Double immunostaining for GFP (green) and NeuN (red) revealed that neurons were specifically transduced (<b>D–O</b>). Merged images (<b>F, I, L, O</b>) also show Hoechst counterstaining. Neurons in many hippocampal sub-regions were transduced including Amon’s horn (<b>D–F</b>) and dentate gyrus (<b>G–I</b>). GFP was observed in Purkinje cells and other neurons in the cerebellum (<b>M–O</b>). Scale bar  = 500 µm (<b>A–C</b>) and 50 µm (<b>D–O</b>).</p

C100 and Aβ protein production <i>in vitro</i> after plasmid transfection.

<p>Analysis of C100 and Aβ protein production <i>in vitro</i> after transfection with plasmids expressing Aβ40-GFP, Aβ42-GFP, C100-GFP or C100^V717F-GFP in cell homogenates (<b>A</b>) and media (<b>B</b>). C100 protein was only detected in cell homogenates after transfection with C100-GFP and C100^V717F-GFP plasmids. No Aβ was detected in cell homogenates after transfection with any plasmid. APP^SWE brain homogenate was used as a positive control for Aβ detection. Strong GFP protein expression indicated successful transfection using all plasmids and β-actin was used as a loading control. Immunoprecipitation of Aβ from the cell culture media revealed Aβ in the media from cells transfected with C100-GFP and C100^V717F-GFP plasmids. Control refers to non-transfected cells.</p

IgG positive cells in the hippocampus and cerebellum.

<p>Mouse IgG positive cells (red) were frequently observed in the hippocampus (<b>B</b>) and cerebellum (<b>C</b>) after injection with AAV2-Aβ40-GFP, AAV2-Aβ42-GFP, AAV2-C100-GFP and AAV2-C100^V717F-GFP in comparison to after AAV2-GFP injection (<b>A</b>). Sections were counterstained with Hoechst (blue). Inserts show distinctive morphology of IgG positive cells. High magnification images of mouse IgG positive cells co-labelled with IBA-1 are shown in <b>D–G</b> (<b>D</b>: IgG staining, <b>E</b>: IBA-1 staining, <b>F</b>: Hoechst, <b>G</b>: merged image). The total number of IgG positive cells was counted in 3 sections per brain in both the hippocampus (<b>H</b>) and cerebellum (<b>I</b>) in the injected hemisphere at 3 and 6 months post-injection. Scale bar  = 200 µm (<b>A–C</b>) and 10 µm (<b>D–G</b>). INJ: injected hemisphere, CONTRA: contralateral hemisphere, ***p<0.001, *p<0.05.</p

Increased mouse IgG staining in the hippocampus and cerebellum.

<p>Example images show normal IgG staining following immunohistochemistry for NeuN after AAV2-GFP injection (<b>A–D</b>) and intense IgG staining after injection of AAV2-Aβ42-GFP (<b>E, G</b>). Increased IgG staining was only observed surrounding the injection site and not in the contralateral hemisphere (<b>F, H</b>). INJ; injected region, CONTRA; contralateral region. Scale bar  = 500 µm.</p