A Cautionary Tale: Endogenous Biotinylated Proteins and Exogenously-Introduced Protein A Cause Antibody-Independent Artefacts in Western Blot Studies of Brain-Derived Proteins.

Antibodies are commonly used to detect or isolate proteins from biological samples. Much attention has been paid to the potential for poorly-characterized antibodies to lead to misleading results, but antibody-independent artefacts may also occur. Here, we recount two examples of antibody-independent artefacts that have confounded the interpretation of results in our search for molecular entities associated with memory loss in Alzheimer's disease (AD). First, when using biotin-avidin systems for antibody detection, endogenous biotinylated proteins created spurious bands in Western blots of brain lysates from AD patients and transgenic mouse models of AD. These artefactual bands occurred in a transgene- and strain-dependent manner. A second, unexpected artefact occurred when Protein A-conjugated Sepharose beads were used to deplete lysates of endogenous immunoglobulins prior to immunopurification of target proteins. In these assays, Protein A shed from the beads, then bound to (and was eluted from) an immunoaffinity matrix designed to capture AD-related proteins. The Protein A then bound detection antibodies when the immunoaffinity eluates were analyzed by Western blot. Both of these artefacts-the endogenous biotinylated proteins and the Protein A artefact-can be monitored by including an "irrelevant" antibody as an experimental control (e.g., running a parallel protocol in which the antibody directed against the target of interest is replaced by a non-specific antibody).

Human cerebrospinal fluid 6E10-immunoreactive protein species contain amyloid precursor protein fragments.

In a previous study, we reported that levels of two types of protein species-a type of ~55-kDa species and a type of ~15-kDa species-are elevated in the lumbar cerebrospinal fluid (CSF) of cognitively intact elderly individuals who are at risk for Alzheimer's disease (AD). These species are immunoreactive to the monoclonal antibody 6E10, which is directed against amino acids 6-10 of amyloid-ß (Aß), and their levels correlate with levels of total tau and tau phosphorylated at Thr181. In this study, we investigated the molecular composition of these AD-related proteins using immunoprecipitation (IP)/Western blotting coupled with IP/mass spectrometry. We show that canonical Aß1-40/42 peptides, together with amyloid-ß precursor protein (APP) fragments located N-terminally of Aß, are present in the ~55-kDa, 6E10-immunoreactive species. We demonstrate that APP fragments located N-terminally of Aß, plus the N-terminal region of Aß, are present in the ~15-kDa, 6E10-immunoreactive species. These findings add to the catalog of AD-related Aß/APP species found in CSF and should motivate further study to determine whether these species may serve as biomarkers of disease progression.

The role of TREM2 in Alzheimer's disease and other neurodegenerative disorders.

Alzheimer's disease is a genetically complex disorder; rare variants in the triggering receptor expressed on myeloid cells 2 (TREM2) gene have been shown to as much as triple an individual's risk of developing Alzheimer's disease. TREM2 is a transmembrane receptor expressed in cells of the myeloid lineage, and its association with Alzheimer's disease supports the involvement of immune and inflammatory pathways in the cause of the disease, rather than as a consequence of the disease. TREM2 variants associated with Alzheimer's disease induce partial loss of function of the TREM2 protein and alter the behaviour of microglial cells, including their response to amyloid plaques. TREM2 variants have also been shown to cause polycystic lipomembranous osteodysplasia with sclerosing leukoencephalopathy and frontotemporal dementia. Although the low frequency of TREM2 variants makes it difficult to establish robust genotype-phenotype correlations, such studies are essential to enable a comprehensive understanding of the role of TREM2 in different neurological diseases, with the ultimate goal of developing novel therapeutic approaches.

Quantitative Comparison of Dense-Core Amyloid Plaque Accumulation in Amyloid-ß Protein Precursor Transgenic Mice.

There exist several dozen lines of transgenic mice that express human amyloid-ß protein precursor (AßPP) with Alzheimer's disease (AD)-linked mutations. AßPP transgenic mouse lines differ in the types and amounts of Aß that they generate and in their spatiotemporal patterns of expression of Aß assemblies, providing a toolkit to study Aß amyloidosis and the influence of Aß aggregation on brain function. More complete quantitative descriptions of the types of Aß assemblies present in transgenic mice and in humans during disease progression should add to our understanding of how Aß toxicity in mice relates to the pathogenesis of AD. Here, we provide a direct quantitative comparison of amyloid plaque burdens and plaque sizes in four lines of AßPP transgenic mice. We measured the fraction of cortex and hippocampus occupied by dense-core plaques, visualized by staining with Thioflavin S, in mice from young adulthood through advanced age. We found that the plaque burdens among the transgenic lines varied by an order of magnitude: at 15 months of age, the oldest age studied, the median cortical plaque burden in 5XFAD mice was already ∼4.5 times that of 21-month-old Tg2576 mice and ∼15 times that of 21-24-month-old rTg9191 mice. Plaque-size distributions changed across the lifespan in a line- and region-dependent manner. We also compared the dense-core plaque burdens in the mice to those measured in a set of pathologically-confirmed AD cases from the Nun Study. Cortical plaque burdens in Tg2576, APPSwePS1ΔE9, and 5XFAD mice eventually far exceeded those measured in the human cohort.

Caspase-2 cleavage of tau reversibly impairs memory.

In Alzheimer's disease (AD) and other tauopathies, the tau protein forms fibrils, which are believed to be neurotoxic. However, fibrillar tau has been dissociated from neuron death and network dysfunction, suggesting the involvement of nonfibrillar species. Here we describe a novel pathological process in which caspase-2 cleavage of tau at Asp314 impairs cognitive and synaptic function in animal and cellular models of tauopathies by promoting the missorting of tau to dendritic spines. The truncation product, Δtau314, resists fibrillation and is present at higher levels in brains from cognitively impaired mice and humans with AD. The expression of tau mutants that resisted caspase-2 cleavage prevented tau from infiltrating spines, dislocating glutamate receptors and impairing synaptic function in cultured neurons, and it prevented memory deficits and neurodegeneration in mice. Decreasing the levels of caspase-2 restored long-term memory in mice that had existing deficits. Our results suggest an overall treatment strategy for re-establishing synaptic function and restoring memory in patients with AD by preventing tau from accumulating in dendritic spines.

Quaternary Structure Defines a Large Class of Amyloid-ß Oligomers Neutralized by Sequestration.

The accumulation of amyloid-ß (Aß) as amyloid fibrils and toxic oligomers is an important step in the development of Alzheimer's disease (AD). However, there are numerous potentially toxic oligomers and little is known about their neurological effects when generated in the living brain. Here we show that Aß oligomers can be assigned to one of at least two classes (type 1 and type 2) based on their temporal, spatial, and structural relationships to amyloid fibrils. The type 2 oligomers are related to amyloid fibrils and represent the majority of oligomers generated in vivo, but they remain confined to the vicinity of amyloid plaques and do not impair cognition at levels relevant to AD. Type 1 oligomers are unrelated to amyloid fibrils and may have greater potential to cause global neural dysfunction in AD because they are dispersed. These results refine our understanding of the pathogenicity of Aß oligomers in vivo.

Characterization of a Novel Mouse Model of Alzheimer's Disease--Amyloid Pathology and Unique ß-Amyloid Oligomer Profile.

Amyloid plaques composed of ß-amyloid (Aß) protein are a pathological hallmark of Alzheimer's disease. We here report the generation and characterization of a novel transgenic mouse model of Aß toxicity. The rTg9191 mice harbor a transgene encoding the 695 amino-acid isoform of human amyloid precursor protein (APP) with the Swedish and London mutations (APPNLI) linked to familial Alzheimer's disease, under the control of a tetracycline-response element, as well as a transgene encoding the tetracycline transactivator, under the control of the promoter for calcium-calmodulin kinase IIα. In these mice, APPNLI is expressed at a level four-fold that of endogenous mouse APP and its expression is restricted to forebrain regions. Transgene expression was suppressed by 87% after two months of doxycycline administration. Histologically, we showed that (1) Aß plaques emerged in cerebral cortex and hippocampus as early as 8 and 10.5-12.5 months of age, respectively; (2) plaque deposition progressed in an age-dependent manner, occupying up to 19% of cortex at ~25 months of age; and (3) neuropathology--such as abnormal neuronal architecture, tau hyperphosphorylation and misfolding, and neuroinflammation--was observed in the vicinity of neuritic plaques. Biochemically, we determined total Aß production at varied ages of mice, and we showed that mice produced primarily fibrillar Aß assemblies recognized by conformation-selective OC antibodies, but few non-fibrillar oligomers (e.g., Aß*56) detectable by A11 antibodies. Finally, we showed that expression of the tetracycline transactivator resulted in reduced brain weight and smaller dentate-gyrus size. Collectively, these data indicate that rTg9191 mice may serve as a model for studying the neurological effects of the fibrillar Aß assemblies in situ.

More than a FAD: the in vivo effects of disease-linked presenilin-1 mutations.

Mutations in presenilins are linked to familial autosomal dominant Alzheimer's disease. In this issue of Neuron, Xia et al. (2015) show that a disease-linked mutation leads to loss of γ-secretase function, cognitive decline, and neurodegeneration when knocked into the mouse genome.

ß-Amyloid oligomers in aging and Alzheimer's disease.

Alzheimer's disease (AD) is a fatal neurodegenerative disorder, and the most common cause of dementia in the elderly. The cause of AD is not known, but genetic evidence strongly supports the hypothesis that pathological aggregation of the ß-amyloid protein (Aß) triggers the disease process. AD has a long preclinical phase, lasting a decade or more. It is during this preclinical phase, before the irreversible neuron loss that characterizes the dementia phase of the disease, that therapies are most likely to be effective. If we are to block AD during the preclinical phase, we must identify the Aß species that are present before there are overt symptoms and that are associated with downstream markers of pathology. A specific soluble Aß assembly, the putative dodecamer "Aß*56," is present in the brains and cerebrospinal fluid of cognitively intact individuals and correlates with markers of synaptic dysfunction and neuronal injury. This assembly also correlates with memory dysfunction in multiple lines of transgenic mice that model the preclinical phase of AD. We suggest that Aß*56 has a critical role during the earliest phase of AD and might serve as a molecular trigger of the disease.

Correlation of specific amyloid-ß oligomers with tau in cerebrospinal fluid from cognitively normal older adults.

IMPORTANCE: To improve the ability to develop treatments that prevent incipient Alzheimer disease (AD) from progressing to overt AD, it is important to understand the molecular basis of the earliest pathophysiological abnormalities and to determine how amyloid-ß (Aß) is involved very early in its pathogenesis. OBJECTIVE: To investigate 2 specific Aß oligomers, Aß trimers and Aß*56, in human cerebrospinal fluid (CSF); evaluate the effects of aging and AD; and obtain support for the hypothesis that they may be pathogenic by determining their relationships to CSF tau. DESIGN: A CSF sampling study. SETTINGS: The University of Minnesota Medical School in Minneapolis, Minnesota, and the Salhgrenska University Hospital, Sweden. PARTICIPANTS: Forty-eight older adults with mild cognitive impairment or AD (impaired group); 49 age-matched cognitively intact control subjects (unimpaired group); and 10 younger, normal control subjects. MAIN OUTCOME MEASURES: Measurements of CSF Aß trimers, Aß*56, the 42-amino acid Aß isoform (Aß1-42), total tau (T-tau), and phospho-tau 181 (p-tau(181)). The hypothesis being tested was formulated after data collection. RESULTS We observed that Aß trimers and Aß*56 levels increased with age; within the unimpaired group, they were elevated in subjects with T-tau/Aß1-42 ratios greater than a cutoff that distinguished the unimpaired group from subjects with AD. In the unimpaired group, T-tau and p-tau(181) were found to correlate strongly with Aß trimers and Aß*56 (r > 0.63), but not with Aß1-42 (-0.10 < r < -0.01). The strong correlations were found to be attenuated in the impaired group. CONCLUSIONS AND RELEVANCE: In cognitively intact older adults, CSF Aß trimers and Aß*56 were elevated in individuals at risk for AD, and they showed stronger relationships with tau than did Aß1-42, a surrogate for Aß fibril deposition. These findings suggest that prior to overt symptoms, 1 or both of the Aß oligomers, but not fibrillar Aß, is coupled to tau; however, this coupling is weakened or broken when AD advances to symptomatic stages. The uncoupling is interesting in light of the failure of experimental Aß therapies to improve mild cognitive impairment/AD, which has prompted a shift in the timing of Aß therapies to asymptomatic subjects. Knowing which Aß species to target in asymptomatic subjects may enhance the success of future treatments for AD.

Frontotemporal degeneration, the next therapeutic frontier: molecules and animal models for frontotemporal degeneration drug development.

Frontotemporal degeneration (FTD) is a common cause of dementia for which there are currently no approved therapies. Over the past decade, there has been an explosion of knowledge about the biology and clinical features of FTD that has identified a number of promising therapeutic targets as well as animal models in which to develop drugs. The close association of some forms of FTD with neuropathological accumulation of tau protein or increased neuroinflammation due to progranulin protein deficiency suggests that a drug's success in treating FTD may predict efficacy in more common diseases such as Alzheimer's disease. A variety of regulatory incentives, clinical features of FTD such as rapid disease progression, and relatively pure molecular pathology suggest that there are advantages to developing drugs for FTD as compared with other more common neurodegenerative diseases such as Alzheimer's disease. In March 2011, the Frontotemporal Degeneration Treatment Study Group sponsored a conference entitled "FTD, the Next Therapeutic Frontier," which focused on preclinical aspects of FTD drug development. The goal of the meeting was to promote collaborations between academic researchers and biotechnology and pharmaceutical researchers to accelerate the development of new treatments for FTD. Here we report the key findings from the conference, including the rationale for FTD drug development; epidemiological, genetic, and neuropathological features of FTD; FTD animal models and how best to use them; and examples of successful drug development collaborations in other neurodegenerative diseases.

The advantages of frontotemporal degeneration drug development (part 2 of frontotemporal degeneration: the next therapeutic frontier).

Frontotemporal degeneration (FTD) encompasses a spectrum of related neurodegenerative disorders with behavioral, language, and motor phenotypes for which there are currently no effective therapies. This is the second of two articles that summarize the presentations and discussions that occurred at two symposia in 2011 sponsored by the Frontotemporal Degeneration Treatment Study Group, a collaborative group of academic and industry researchers that is devoted to developing treatments for FTD. This article discusses the current status of FTD clinical research that is relevant to the conduct of clinical trials, and why FTD research may be an attractive pathway for developing therapies for neurodegenerative disorders. The clinical and molecular features of FTD, including rapid disease progression and relatively pure molecular pathology, suggest that there are advantages to developing drugs for FTD as compared with other dementias. FTD qualifies as orphan indication, providing additional advantages for drug development. Two recent sets of consensus diagnostic criteria will facilitate the identification of patients with FTD, and a variety of neuropsychological, functional, and behavioral scales have been shown to be sensitive to disease progression. Moreover, quantitative neuroimaging measurements demonstrate progressive brain atrophy in FTD at rates that may surpass Alzheimer's disease. Finally, the similarities between FTD and other neurodegenerative diseases with drug development efforts already underway suggest that FTD researchers will be able to draw on this experience to create a road map for FTD drug development. We conclude that FTD research has reached sufficient maturity to pursue clinical development of specific FTD therapies.

Grape seed polyphenolic extract specifically decreases aß*56 in the brains of Tg2576 mice.

Amyloid-ß (Aß) oligomers, found in the brains of Alzheimer's disease (AD) patients and transgenic mouse models of AD, cause synaptotoxicity and memory impairment. Grape seed polyphenolic extract (GSPE) inhibits Aß oligomerization in vitro and attenuates cognitive impairment and AD-related neuropathology in the brains of transgenic mice. In the current study, GSPE was administered to Tg2576 mice for a period of five months. Treatment significantly decreased brain levels of Aß*56, a 56-kDa Aß oligomer previously shown to induce memory dysfunction in rodents, without changing the levels of transgenic amyloid-ß protein precursor, monomeric Aß, or other Aß oligomers. These results thus provide the first demonstration that a safe and affordable intervention can lower the levels of a memory-impairing Aß oligomer in vivo and strongly suggest that GSPE should be further tested as a potential prevention and/or therapy for AD.

'Too much good news' - are Alzheimer mouse models trying to tell us how to prevent, not cure, Alzheimer's disease?

Scores of compounds ameliorate cognitive deficits or neuropathology in transgenic mouse models of Alzheimer's disease (AD), yet these triumphs in mice have not translated into successful therapies for people. Why have studies in mice failed to predict results of human trials? We argue that most transgenic mouse 'models of AD' actually simulate the asymptomatic phase of the disease, and the results of interventional studies in these mice should be considered in the context of disease prevention. In addition, recent advances in imaging technology and biomarker discovery should aid in comparisons of mouse and human neurological status and, importantly, might allow us to predict better the response of people to drugs tested in mice.

Probing the biology of Alzheimer's disease in mice.

Alzheimer's disease (AD), the most common cause of dementia among the elderly, may either represent the far end of a continuum that begins with age-related memory decline or a distinct pathobiological process. Although mice that faithfully model all aspects of AD do not yet exist, current mouse models have provided valuable insights into specific aspects of AD pathogenesis. We will argue that transgenic mice expressing amyloid precursor protein should be considered models of accelerated brain aging or asymptomatic AD, and the results of interventional studies in these mice should be considered in the context of primary prevention. Studies in mice have pointed to the roles of soluble beta-amyloid (Abeta) oligomers and soluble tau in disease pathogenesis and support a model in which soluble Abeta oligomers trigger synaptic dysfunction, but formation of abnormal tau species leads to neuron death and cognitive decline severe enough to warrant a dementia diagnosis.

Gap junctional coupling and connexin immunoreactivity in rabbit retinal glia.

Gap junctions provide a pathway for the direct intercellular exchange of ions and small signaling molecules. Gap junctional coupling between retinal astrocytes and between astrocytes and Müller cells, the principal glia of vertebrate retinas, has been previously demonstrated by the intercellular transfer of gap-junction permeant tracers. However, functional gap junctions have yet to be demonstrated between mammalian Müller cells. In the present study, when the gap-junction permeant tracers Neurobiotin and Lucifer yellow were injected into a Müller cell via a patch pipette, the tracers transferred to at least one additional cell in more than half of the cases examined. Simultaneous whole-cell recordings from pairs of Müller cells in the isolated rabbit retina revealed electrical coupling between closely neighboring cells, confirming the presence of functional gap junctions between rabbit Müller cells. The limited degree of this coupling suggests that Müller cell-Müller cell gap junctions may coordinate the functions of small ensembles of these glial cells. Immunohistochemistry and immunoblotting were used to identify the connexins in rabbit retinal glia. Connexin30 (Cx30) and connexin43 (Cx43) immunoreactivities were associated with astrocytes in the medullary ray region of the retinas of both pigmented and albino rabbits. Connexin43 was also found in Müller cells, but antibody recognition differed between astrocytic and Müller cell connexin43.

D-serine and serine racemase are present in the vertebrate retina and contribute to the physiological activation of NMDA receptors.

d-serine has been proposed as an endogenous modulator of N-methyl-d-aspartate (NMDA) receptors in many brain regions, but its presence and function in the vertebrate retina have not been characterized. We have detected d-serine and its synthesizing enzyme, serine racemase, in the retinas of several vertebrate species, including salamanders, rats, and mice and have localized both constituents to Müller cells and astrocytes, the two major glial cell types in the retina. Physiological studies in rats and salamanders demonstrated that, in retinal ganglion cells, d-serine can enhance excitatory currents elicited by the application of NMDA, as well as the NMDA receptor component of light-evoked synaptic responses. Application of d-amino acid oxidase, which degrades d-serine, reduced the magnitude of NMDA receptor-mediated currents, raising the possibility that endogenous d-serine serves as a ligand for setting the sensitivity of NMDA receptors under physiological conditions. These observations raise exciting new questions about the role of glial cells in regulating the excitability of neurons through release of d-serine.

Connexin immunoreactivity in glial cells of the rat retina.

The rat retina contains two types of macroglial cells, Müller cells, radial glial cells that are the principal macroglial cells of vertebrate retinas, and astrocytes associated with the surface vasculature. In addition to the often-described gap-junctional coupling between astrocytes, coupling also occurs between astrocytes and Müller cells. Immunohistochemistry and confocal microscopy were used to identify connexins in the retinas of pigmented rats. Several antibodies directed against connexin43 stained astrocytes, identified using antibodies directed against glial fibrillary acidic protein (GFAP). In addition, two connexin43 antibodies stained Müller cells, identified with antibodies directed against S100 or glutamine synthetase. Connexin30-immunoreactive puncta were confined to the vitreal surface of the retina and colocalized with GFAP-immunoreactive astrocyte processes. Connexin45 immunoreactivity was associated with both astrocytes and Müller cells. We conclude that retinal glial cells express multiple connexins, and the patterns of immunostaining that we observe in this study are consistent with the expression of connexins30, -43, and possibly -45 by astrocytes and the expression of connexins43 and -45 by Müller cells. As gap-junction channels may be formed by both homotypic and heterotypic hemichannels, and the hemichannels may themselves be homomeric or heteromeric, there exists a multitude of possible gap-junction channels that could underlie the homotypic coupling between retinal astrocytes and the heterotypic coupling between astrocytes and Müller cells.