Questions concerning the role of amyloid-β in the definition, aetiology and diagnosis of Alzheimer's disease

The dominant hypothesis of Alzheimer’s disease (AD) aetiology, the neuropathological guidelines for diagnosing AD and the majority of high-profile therapeutic efforts, in both research and in clinical practice, have been built around one possible causal factor, amyloid-β (Aβ). However, the causal link between Aβ and AD remains unproven. Here, in the context of a detailed assessment of historical and contemporary studies, we raise critical questions regarding the role of Aβ in the definition, diagnosis and aetiology of AD. We illustrate that a holistic view of the available data does not support an unequivocal conclusion that Aβ has a central or unique role in AD. Instead, the data suggest alternative views of AD aetiology are potentially valid, at this time. We propose that an unbiased way forward for the field, beyond the current Aβ-centric approach, without excluding a role for Aβ, is required to come to an accurate understanding of AD dementia and, ultimately, an effective treatment.This work was supported by the Boyarsky family, Battersby family, David King and family

Quantification of AMPA receptor subunits and RNA editing-related proteins in the J20 mouse model of Alzheimer’s disease by capillary western blotting

IntroductionAccurate modelling of molecular changes in Alzheimer’s disease (AD) dementia is crucial for understanding the mechanisms driving neuronal pathology and for developing treatments. Synaptic dysfunction has long been implicated as a mechanism underpinning memory dysfunction in AD and may result in part from changes in adenosine deaminase acting on RNA (ADAR) mediated RNA editing of the GluA2 subunit of AMPA receptors and changes in AMPA receptor function at the post synaptic cleft. However, few studies have investigated changes in proteins which influence RNA editing and notably, AD studies that focus on studying changes in protein expression, rather than changes in mRNA, often use traditional western blotting.MethodsHere, we demonstrate the value of automated capillary western blotting to investigate the protein expression of AMPA receptor subunits (GluA1-4), the ADAR RNA editing proteins (ADAR1-3), and proteins known to regulate RNA editing (PIN1, WWP2, FXR1P, and CREB1), in the J20 AD mouse model. We describe extensive optimisation and validation of the automated capillary western blotting method, demonstrating the use of total protein to normalise protein load, in addition to characterising the optimal protein/antibody concentrations to ensure accurate protein quantification. Following this, we assessed changes in proteins of interest in the hippocampus of 44-week-old J20 AD mice.ResultsWe observed an increase in the expression of ADAR1 p110 and GluA3 and a decrease in ADAR2 in the hippocampus of 44-week-old J20 mice. These changes signify a shift in the balance of proteins that play a critical role at the synapse. Regression analysis revealed unique J20-specific correlations between changes in AMPA receptor subunits, ADAR enzymes, and proteins that regulate ADAR stability in J20 mice, highlighting potential mechanisms mediating RNA-editing changes found in AD.DiscussionOur findings in J20 mice generally reflect changes seen in the human AD brain. This study underlines the importance of novel techniques, like automated capillary western blotting, to assess protein expression in AD. It also provides further evidence to support the hypothesis that a dysregulation in RNA editing-related proteins may play a role in the initiation and/or progression of AD

Neuroinflammation and Neuronal Loss Precede Aβ Plaque Deposition in the hAPP-J20 Mouse Model of Alzheimer’s Disease

Recent human trials of treatments for Alzheimer's disease (AD) have been largely unsuccessful, raising the idea that treatment may need to be started earlier in the disease, well before cognitive symptoms appear. An early marker of AD pathology is therefore needed and it is debated as to whether amyloid-βAβ? plaque load may serve this purpose. We investigated this in the hAPP-J20 AD mouse model by studying disease pathology at 6, 12, 24 and 36 weeks. Using robust stereological methods, we found there is no neuron loss in the hippocampal CA3 region at any age. However loss of neurons from the hippocampal CA1 region begins as early as 12 weeks of age. The extent of neuron loss increases with age, correlating with the number of activated microglia. Gliosis was also present, but plateaued during aging. Increased hyperactivity and spatial memory deficits occurred at 16 and 24 weeks. Meanwhile, the appearance of plaques and oligomeric Aβ were essentially the last pathological changes, with significant changes only observed at 36 weeks of age. This is surprising given that the hAPP-J20 AD mouse model is engineered to over-expresses Aβ. Our data raises the possibility that plaque load may not be the best marker for early AD and suggests that activated microglia could be a valuable marker to track disease progression.Funding provided by Iain S. Gray Foundation, Stanley and John Roth, Patricia A. Quick foundation, David King, Doug Battersby, Tony and Vivian Howland-Rose, Walter and Edith Sheldon, Gleneagle Securities, Bill Gruy, Geoffrey Towner, Amadeus Energy Ltd., Nick and Melanie Kell, J. O. and J. R. Wicking Trust and the Mason Foundation, the New South Wales Government, through their office for Science and Medical Research, and SpinalCure Australia. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

A Conditional Deletion of the NR1 Subunit of the NMDA Receptor in Adult Spinal Cord Dorsal Horn Reduces NMDA Currents and Injury-Induced Pain

To determine the importance of the NMDA receptor (NMDAR) in pain hypersensitivity after injury, the NMDAR1 (NR1) subunit was selectively deleted in the lumbar spinal cord of adult mice by the localized injection of an adenoassociated virus expressing Cre recombinase into floxed NR1 mice. NR1 subunit mRNA and dendritic protein are reduced by 80% in the area of the virus injection, and NMDA currents, but not AMPA currents, are reduced 86–88% in lamina II neurons. The spatial NR1 knock-out does not alter heat or cold paw-withdrawal latencies, mechanical threshold, or motor function. However, injury-induced pain produced by intraplantar formalin is reduced by 70%. Our results demonstrate conclusively that the postsynaptic NR1 receptor subunit in the lumbar dorsal horn of the spinal cord is required for central sensitization, the central facilitation of pain transmission produced by peripheral injury

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The meteorology of cytokine storms, and the clinical usefulness of this knowledge

The term cytokine storm has become a popular descriptor of the dramatic harmful consequences of the rapid release of polypeptide mediators, or cytokines, that generate inflammatory responses. This occurs throughout the body in both non-infectious and infectious disease states, including the central nervous system. In infectious disease it has become a useful concept through which to appreciate that most infectious disease is not caused directly by a pathogen, but by an overexuberant innate immune response by the host to its presence. It is less widely known that in addition to these roles in disease pathogenesis these same cytokines are also the basis of innate immunity, and in lower concentrations have many essential physiological roles. Here we update this field, including what can be learned through the history of how these interlinking three aspects of biology and disease came to be appreciated. We argue that understanding cytokine storms in their various degrees of acuteness, severity and persistence is essential in order to grasp the pathophysiology of many diseases, and thus the basis of newer therapeutic approaches to treating them. This particularly applies to the neurodegenerative diseases

A Neurologist’s Guide to TNF Biology and to the Principles behind the Therapeutic Removal of Excess TNF in Disease

Tumor necrosis factor (TNF) is an ancient and widespread cytokine required in small amounts for much physiological function. Higher concentrations are central to innate immunity, but if unchecked this cytokine orchestrates much chronic and acute disease, both infectious and noninfectious. While being a major proinflammatory cytokine, it also controls homeostasis and plasticity in physiological circumstances. For the last decade or so these principles have been shown to apply to the central nervous system as well as the rest of the body. Nevertheless, whereas this approach has been a major success in treating noncerebral disease, its investigation and potential widespread adoption in chronic neurological conditions has inexplicably stalled since the first open trial almost a decade ago. While neuroscience is closely involved with this approach, clinical neurology appears to be reticent in engaging with what it offers patients. Unfortunately, the basic biology of TNF and its relevance to disease is largely outside the traditions of neurology. The purpose of this review is to facilitate lowering communication barriers between the traditional anatomically based medical specialties through recognition of shared disease mechanisms and thus advance the prospects of a large group of patients with neurodegenerative conditions for whom at present little can be done