Alzheimer Research Forum on the web

Kinoshita and Clark announce Alzforum, an online community for Alzheimer’s disease (AD) researchers:

Alzforum: E-Science for Alzheimer Disease

The Alzheimer Research Forum Web site (http://www.alzforum.org) is an independent research project to develop an online community resource to manage scientific knowledge, information, and data about Alzheimer disease (AD). Its goals are to promote rapid communication, research efficiency, and collaborative, multidisciplinary interactions. Introducing new knowledge management approaches to AD research has a potentially large societal value. … In addition to imposing a heavy burden on family caregivers and society at large, AD and related neurodegenerative disorders are among the most complex and challenging in biomedicine. Researchers have produced an abundance of data implicating diverse biological mechanisms. Important factors include genes, environmental risks, changes in cell functions, DNA damage, accumulation of misfolded proteins, cell death, immune responses, changes related to aging, and reduced regenerative capacity. Yet there is no agreement on the fundamental causes of AD. The situations regarding Parkinson, Huntington, and amyotrophic lateral sclerosis (ALS) are similar. The challenge of integrating so much data into testable hypotheses and unified concepts is formidable. What is more, basic understanding of these diseases needs to intersect with an equally complex universe of pharmacology, medicinal chemistry, animal studies, and clinical trials. In this chapter, we will describe the approaches developed by Alzforum to achieve knowledge integration through information technology and virtual community-building. We will also propose some future directions in the application of Web-based knowledge management systems in neuromedicine.

It’s an ambitious mission and could use the support of the community, beginning with your participation.

 

Mitochondria aren’t always to blame

Two recent reviews discuss the evidence that mitochondria (specifically, age- and damage-related dysfunction in these organelles) are responsible for age-related degenerative conditions. Both reviews focus on oxidative stress as a primary mechanism underlying the connections, but depending on the disease in question, they reach rather different conclusions about the significance of mitochondrial damage.

Kim et al. describe the role for mitochondria (in particular, reactive oxygen species [ROS] produced by damaged mitos and the concomitant inflammation) in the etiology of late-life insulin resistance. The authors conclude that the balance of evidence supports a role for mitochondrial damage in late-onset diabetes, and I think their arguments are reasonable (though I’m hard pressed to think of a phenomenon that doesn’t play some role in diabetes).

Meanwhile, Fukui and Moraes critically evaluate the idea that ROS-induced damage causing further ROS production, and the possibility that such a “vicious cycle” could participate in the development of neurodegenerative diseases. Their conclusion is that the field has been led astray by results of in vitro experiments that don’t accurately model the situation in vivo.

 

Proteasome inhibition and the mitochondria

The proteasome is an essential agent in cellular protein turnover, recognizing and targeting polyubiquitinated proteins for degradation — a process that serves both a regenerative function (by turning proteins back into amino acids, the raw materials for proteins yet to be synthesized) and a regulatory one (a protein that has been degraded can no longer act within the cell).

Papa and Rockwell report that reversible proteasome inhibition has long-term effects on the mitochondria, causing diminished energy production and increased generation of reactive oxygen species (ROS) in neurons. This increase in oxidative stress, in turn, results in increased cell death via apoptosis:

Persistent mitochondrial dysfunction and oxidative stress hinder neuronal cell recovery from reversible proteasome inhibition

Oxidative stress, proteasome impairment and mitochondrial dysfunction are implicated as contributors to ageing and neurodegeneration. Using mouse neuronal cells, we showed previously that the reversible proteasome inhibitor, [N-benzyloxycarbonyl-Ile-Glu (O-t-bytul)-Ala-leucinal; (PSI)] induced excessive reactive oxygen species (ROS) that mediated mitochondrial damage and a caspase-independent cell death. Herein, we examined whether this insult persists in neuronal cells recovering from inhibitor removal over time. Recovery from proteasome inhibition showed a time and dose-dependent cell death that was accompanied by ROS overproduction, caspase activation and mitochondrial membrane permeabilization with the subcellular relocalizations of the proapoptotic proteins, Bax, cytochrome c and the apoptosis inducing factor (AIF). Caspase inhibition failed to promote survival indicating that cell death was caspase-independent. Treatments with the antioxidant N-acetyl-cysteine (NAC) were needed to promote survival in cell recovering from mild proteasome inhibition while overexpression of the antiapoptotic protein Bcl-xL together with NAC attenuated cell death during recovery from potent inhibition. Whereas inhibitor removal increased proteasome function, cells recovering from potent proteasome inhibition showed excessive levels of ubiquitinated proteins that required the presence of NAC for their removal. Collectively, these results suggest that the oxidative stress and mitochondrial inhibition induced by proteasome inhibition persists to influence neuronal cell survival when proteasome function is restored.

Here’s what caught my eye about this paper: Mitochondrial ROS production is widely considered to play a causative role in cellular aging. Mitochondria, in turn, accumulate damage over the lifespan, which causes an acceleration in ROS production — a nasty positive-feedback loop. Based on these findings, however, I wonder whether the decline in mitochondrial function could be driven not only from within (by oxidative damage to mitos causing further oxidation), but also from without: We know that proteasome efficiency also declines during aging; it would seem likely that this functional decline could further erode mitochondrial function.

One corollary of this hypothesis is that even if we were able to completely eliminate oxidative damage, we’d still suffer diminution in mitochondrial capacity as a result of proteasomal decline –unless, of course, this decline and its effect on mitochondria operate via a non-oxidative mechanism, in which case eliminating oxidative damage would kill two birds with one stone.

 

Mitochondrial dysfunction in Alzheimer’s

The brains of Alzheimer’s disease (AD) patients are riddled with plaques of amyloid-beta (Aß) protein — but what causes the accumulation of the plaques?

One candidate is mitochondrial dysfunction, which can result in high levels of reactive oxygen species (ROS) and also rob the cell of energy it needs for maintenance of homeostasis. Hauptmann et al. report that in an AD mouse model, mitochondrial dysfunction can be observed very early in the progression of the disease: Indeed, mitochondria begin to exhibit respiratory-chain defects well before extracellular plaques can be observed:

Mitochondrial dysfunction: An early event in Alzheimer pathology accumulates with age in AD transgenic mice

Recent evidence suggests mitochondrial dysfunction as a common early pathomechanism in Alzheimer’s disease integrating genetic factors related to enhanced amyloid-beta (Aß) production and tau-hyperphosphorylation with aging, as the most relevant sporadic risk factor. To further clarify the synergistic effects of aging and Aß pathology, we used isolated mitochondria of double Swedish and London mutant APP transgenic mice and of non-tg littermates. Pronounced mitochondrial dysfunction in adult Thy-1 APP mice, such as a drop of mitochondrial membrane potential and reduced ATP-levels already appeared at 3 months when elevated intracellular but not extracellular Aß deposits are present. Mitochondrial dysfunction was associated with higher levels of reactive oxygen species, an altered Bcl-xL/Bax ratio and reduction of COX IV activity. We observed significant decreases in state 3 respiration and FCCP-uncoupled respiration in non-tg mice after treatment with extracellular Aß. Similar deficits were seen only in aged Thy-1 APP mice, probably due to compensation within the respiratory chain in young animals. We conclude that Aß dependent mitochondrial dysfunction starts already at 3 months in this AD model before extracellular deposition of Aß and progression accelerates substantially with aging.

To the extent that mitochondrial dysfunction contributes to pathology, it could partially explain why AD is a disease of old age, since mitochondria deteriorate over the course of aging.

Granted, these mice are APP mutants and already primed to develop AD. In a wildtype animal, could mitochondrial dysfunction somehow interfere with protein folding, secretion or clearance machinery and thereby jump-start the process of Aß aggregation?

 

Autophagy, stress resistance, and aging

The cellular housekeeping program known as autophagy — which allows the recycling of old (and potentially damaged) proteins, lipids, and even whole organelles — is essential to maintaining cellular health over long periods of time. Recent findings implicate autophagy in the regulation of lifespan: Without autophagy, damaged macromolecules will slowly accumulate over time, potentially resulting in a breakdown of cellular homeostasis termed a garbage catastrophe — and unfortunately, it appears that the efficiency of autophagy decreases with age.

Two recent articles underscore the importance of autophagy in the longevity of two favorite model systems: the fly Drosophila and the worm C. elegans. Juhász and Neufeld report that fly mutants in the autophagy gene Atg7 are viable and develop normally, but are hypersensitive to oxidation and starvation stress (two hallmarks of premature aging mutants) and undergo premature neuronal cell death. These results are consistent with earlier findings linking defects in lysosomal trafficking (the intracellular sorting mechanism that guides the targets of autophagy to their ultimate fate) to shortened longevity, stress resistance and phenotypes of premature aging.

Meanwhile, in the worm (where, autophagy is a two-edged sword, at least in the “brain”) Tóth et al. describe how multiple longevity assurance pathways (IGF-1 and TOR) converge on autophagy. They argue that the regulation of autophagy represents a key mechanism by which these pathways regulate longevity — a model that makes a large number of strong predictions about genetic interactions between these pathways (predictions which, one hopes, the authors are currently hard at work testing).

 

The big picture: Alzheimer’s transcriptome, insulin phosphoproteome

For all you genomics and systems-level junkies out there, here are two very juicy genome-scale (in one case, proteome-scale) studies of two very different aging-related phenomena: From Miller et al., we have a systems-level analysis of gene expression in both Alzheimer’s disease (AD) and normal aging. The authors use the transcriptional data in conjunction with genome annotation to identify pathways that are coherently regulated, either by neurodegenerative changes or age-related decline. Since the authors focus on brain tissue rather than the more easily accessible (or biopsy-able) parts of the body, these findings will be more relevant to understanding the pathology of AD than to its diagnosis. Hence, this approach is complementary to recent studies aimed at identifying proteins that are differentially expressed in the blood of AD patients and can therefore be used as diagnostic biomarkers for the disease.

Meanwhile, Krüger et al. have used mass spectrometry to characterize the tyrosine phosphoproteome of the insulin signaling pathway — in other words, they looked for proteins that are differentially tyrosine-phosphorylated as a result of insulin action. In addition to rounding up proteins already known to be involved, they also report the identification of several novel effectors of insulin signaling. The technique appears quite robust, and I look forward to seeing this methodology extended to other aging-related signaling networks (such as the closely related IGF-1 pathway).

 

A new wrinkle for green tea

The potential health benefits of green tea have been widely discussed in the press as well as the scholarly literature (e.g., see our earlier post on the ability of green tea-derived compounds to delay neurodegeneration). Add one more to the list: the green tea component epigallocatechin gallate prevents activation of collagen-degrading proteases in response to UV irradiation. This might in turn help prevent skin wrinkling, a consequence of protease action that is one of the most outwardly visible signs of aging.

Careful readers will remember a similarity between this report and another recent paper about a plant-derived compound: the plant alkaloid berberine has a similar effect on UV-irradiated skin cells. We know that senescent fibroblasts are a major source of matrix metalloproteases following DNA damage. Just as with the earlier paper, the important question is whether these plant-derived molecules are preventing activation of the tumor-suppressive senescence pathway (and therefore risking tumorigenesis later in life), or instead preventing senescent cells from engaging in a deleterious secretory program that damages the tissue microenvironment for no obvious good reason.

 

Micro-RNAs and neurodegeneration

Micro-RNAs are a hot topic in almost every field of molecular biology right now; we’ve discussed them before here in the context of buffering age-related changes in gene expression. These novel regulatory molecules may also play a critical role in neurodegenerative disease, as described in this conference proceedings review by Pete Nelson and co-workers:

MicroRNAs (miRNAs) in Neurodegenerative Diseases

Aging-related neurodegenerative diseases (NDs) are the culmination of many different genetic and environmental influences. Prior studies have shown that RNAs are pathologically altered during the inexorable course of some NDs. Recent evidence suggests that microRNAs (miRNAs) may be a contributing factor in neurodegeneration. miRNAs are brain-enriched, small (22 nucleotides) non-coding RNAs that participate in mRNA translational regulation. Although discovered in the framework of worm development, miRNAs are now appreciated to play a dynamic role in many mammalian brain-related biochemical pathways, including neuroplasticity and stress responses. Research about miRNAs in the context of neurodegeneration is accumulating rapidly, and the goal of this review is to provide perspective for these new data that may be helpful to specialists in either field. An overview is provided about the normal functions for miRNAs, including some of the newer concepts related to the human brain. Recently published studies pertaining to the roles of miRNAs in NDs––including Alzheimer’s disease, Parkinson’s disease and triplet repeat disorders—are described. Finally, a discussion is included with theoretical syntheses and possible future directions in exploring the nexus between miRNA and ND research.

 

Telomerase-independent cytoprotection by TERT

Telomerase is a ribonucleoprotein: the RNA component (TERC) provides the template information, and the protein component (TERT in humans) catalyzes polymerization of new telomeric DNA. Both are essential to the activity of the enzyme — at least, insofar as production of telomeres is concerned. Lee et al. report that TERT alone protects cells from multiple types of potentially lethal stresses, even in the absence of TERC:

TERT promotes cellular and organismal survival independently of telomerase activity

The expression level of the telomerase catalytic subunit (telomerase reverse transcriptase, TERT) positively correlates with cell survival after exposure to several lethal stresses. However, whether the protective role of TERT is independent of telomerase activity has not yet been clearly explored. Here, we genetically evaluated the protective roles of both TERT and telomerase activity against cell death induced by staurosporine (STS) and N-methyl-d-aspartic acid (NMDA). First generation (G1) TERT-deficient mouse embryonic fibroblasts (MEFs) displayed an increased sensitivity to STS, while TERT transgenic MEFs were more resistant to STS-induced apoptosis than wild-type. Deletion of the telomerase RNA component (TERC) failed to alter the sensitivity of TERT transgenic MEFs to STS treatment. Similarly, NMDA-induced excitotoxic cell death of primary neurons was suppressed by TERT, but not by TERC both in vitro and in vivo. Specifically, NMDA accelerated death of TERT-deficient mice, while TERT transgenic mice showed enhanced survival when compared with wild-type littermates after administration of NMDA. In addition, the transgenic expression of TERT protected motor neurons from apoptosis induced by sciatic nerve axotomy. These results indicate that telomerase activity is not essential for the protective function of TERT. This telomerase activity-independent TERT function may contribute to cancer development and aging independently of telomere lengthening.

Note that two of the cell types studied (primary and motor neurons) are entirely post-mitotic — which underscores the novelty of TERT’s new function (though it’s certainly possible that a catalytic function of telomerase could play a role in a postmitotic lineage). For more on the subject of excitotoxic cell death see Robert Sapolsky’s old chestnut Stress, the Aging Brain, and the Mechanisms of Neuron Death.

The obvious next step is to “bash” the TERT protein, introducing target mutations and asking which parts of the protein one can break and still preserve the cytoprotective function. Will the critical region map to the catalytic center? Given the TERC-independence of the phenomenon, it’s doubtful, but stranger things have happened.

 

A genetic screen for Parkinson’s susceptibility

Why are some individuals more susceptible than others to age-related neurodegeneration, such as Parkinson’s disease (PD)? Many major players have been identified: we now know, for example, that misfolding and aggregation of the alpha-synuclein protein contributes substantially to the degeneration of dopaminergic neurons. Genomic amplification of the alpha-synuclein gene contributes to increased risk of PD, probably because more protein means more chances for aggregates to form — the world’s worst lottery.

Most PD patients, however, do not exhibit structural polymorphisms at the alpha-synuclein locus — but they are still likely to possess genetic variants that increase their vulnerability to the disease. These variations run in families, but the genetics are abominably hard: each variant may contribute only a tiny amount to disease risk, but risk is hard to measure — we can only really be sure whether or not a given individual has developed the disease. If we’re lucky enough to have a large pedigree to analyze, we might be able to back-calculate the risk contributions of individual loci, but (a) this is screamingly hard, and (b) the analysis is confounded by the incidence of sporadic disease (i.e., PD that occurs for some reason other than genetic factors).

Therefore, classical genetics in model organisms still holds tremendous value as a means of identifying susceptibility loci for PD and other late-life degenerative illnesses. Hamamichi et al. have performed a screen for worm mutants that misfold alpha-synuclein, and identified 20 genes involved in this process; they infer that the wildtype genes protect against alpha-synuclein aggregation and thereby against degeneration of dopaminergic neurons.

Because the worm allows for full knockdown of gene function (here as a result of RNA interference), the authors were able to observe clear phenotypes rather than the small increases in risk that would result from minor functional variations between polymorphic alleles. Consequently, they were able to identify genes involved in alpha-synuclein processing that would have been difficult or impossible to catch in studies of human populations. The human homologs of the worm genes will be excellent candidates for PD susceptibility loci in the human population, as well as targets for pharmacological intervention.