Model Organisms

Nematode worm

The first model organism that we will address is the nematodeCaenorhabditis elegans.As an inver- tebrate,C. elegansmay seem to have little comparative power to the biology of the human organ- ism. However, its cellular complexity is similar to mammalian systems.⁴²Therefore, if the disease 64 Eric B. Emmons, Youngcho Kim, and Nandakumar S. Narayanan

that is being modeled inC. elegansshares traits on the cellular level to its corresponding form in humans, valuable inferences and generalizations can be made.⁴²

One powerful feature ofC. elegansis its relatively simple, well-characterized genome. Genetic findings inC. eleganscan have great relevance for human disease and have contributed to discov- eries in oncogenetics, metabolism, and neurodegeneration. Another distinct advantage for neuro- science research is that the nematode has just over 300 neurons with well-mapped connections.⁴³ Through the use of anin silicoanalysis of theC. elegansgenome, Kuwabara and O’Neil found that 78% of human genes associated with metabolic deficits had homologues in the nematode genome.⁴⁴The search for genetic factors of aging has been a primary driver in recent years and is increasingly promising as techniques and technologies improve. One line of research utilizes mutations in an effort to find genes associated with lifespan. Candidate genes have been found that both increase and decrease life expectancy inC. elegans. The first“longevity”gene discovered in the nematode wasage-1, which is tied to changes in fertility. It was found to increase average life- span by 40% and maximal lifespan by up to 60%.⁴⁵In a 1997 review, Finch and Tanzi summarize similar mutations that have been found to increase lifespan by 40–100%.⁴⁶These mutations also provide greater protection from exposure to extreme temperatures, free radicals, and UV radia- tion.⁴⁶In contrast, mutations that accelerate the aging process have also been discovered. Knock- out of a mitochondrial heat-shock protein that is important for energy production inC. elegans reduced lifespan and produced symptoms of progeria.⁴⁷

Another important direction of aging research in theC. elegansmodel is toxin accumulation and its related damages. As stated above, the predominant toxin-related theory, oxidative stress, asserts that by-products of mitochondrial respiration reduce cellular integrity. Given harsh environmental conditions, C. elegans can move into an alternative larval phase which can last for three to six months, greatly extending their lifespan.⁴⁸This phase, called the“dauer”stage, provides a fasci- nating illustration of how environmental toxins influence aging. There are two changes in the dauer stage which support oxidative stress theory: (1) the electron transport chain seems to be sup- plemented by alternative energy pathways, and (2) oxygen consumption goes down fourfold as compared to the corresponding larval stage.^49,50Proteotoxicity, the toxic effects of aberrant pro- teins, is another promising direction of research in theC. elegansmodel. In recent work using a heat-shock model, Prahlad and Morimoto found neuronal control to be critical for the response to protein aggregation and determining whether the damage is chronic or acute.^51,52For instance, deleting thermosensory neurons alters the heat-shock response in unconnected neurons, presum- ably through circuit mechanisms involving serotonin.⁵³These signaling mechanisms can directly contribute to neurodegeneration and could contribute to the spread of degeneration throughout the neuraxis.

Theclk-1mutant is a long-lived strain ofC. elegansthat exhibits altered cellular respiration.⁵⁴ Theclk-1gene has been hypothesized to be involved in regulation of the biological clock, affecting the timing of processes ranging from defecation to the cell cycle.⁵⁵It has further been linked to longevity, as mutations in the gene extend lifespan while overexpression reduces lifespan.⁵⁴ Another mutation that reduces lifespan due to toxicity is themev-1strain. It appears that modifica- tions to this gene result in disruption to the electron transport chain, likely causing superoxide buildup and heightened oxidative stress.⁵⁶

Because of the relative ease of genetic modifications in theC. elegansmodel, the nematode has been used to study the genetic foundations of PD. One prime focus of PD research has been on α-synuclein, a gene that encodes theα-synuclein protein. This presynaptic protein is likely involved in cellular trafficking.⁵⁷Importantly, misfolded forms of the protein seem to be related to cell death, specifically destruction of dopaminergic neurons.⁵⁸Of greatest interest to PD,α-synuclein is the primary component of Lewy bodies, one of the classic hallmarks of Parkinson’s pathology.⁵⁹ Various studies have attempted to modifyα-synuclein inC. elegans. Van Ham et al. found that overexpressing humanα-synuclein resulted in the age-dependent accumulation of one form of the protein.⁶⁰ One study overexpressed α-synuclein in dopaminergic neurons, observed 65 Animal Models of Pathological Aging

degeneration of those cells, and found behavioral modifications associated with that change.⁶¹Another promising direction for Parkinson’s research inC. elegansis overexpression of the leucine-rich repeat kinase 2 (LRRK2) protein. This has been shown to produce selective degeneration of dopaminergic neurons, suggesting that it could be an important part of the puzzle.⁶²However, some caveats to the study of PD inC. elegansbear mentioning. For one, LRRK2 overexpression inC. elegansresults in neuronal degeneration but does not lead toα-synuclein inclusions because the species doesn’t have endogenous forms of the protein.⁶³Furthermore, whileα-synuclein-related degeneration can be tar- geted to dopaminergic neurons, it doesn’t seem to be progressive as it is in human forms of PD.⁶⁴ Finally, it appears that only 10–20% of PD cases are genetically based.⁶³In summary, genetic research into PD in the worm has already produced valuable insights into the characterization of the disease across the spectrum. Still, generalizing insights from nematodes to humans remains challenging.

Fewer studies have usedC. elegansto study AD. One promising direction involves studying the hallmark aggregation of proteins into NFTs. The primary component of NFTs, the tau protein, is particularly characteristic. Indeed, AD and similar disorders are sometimes referred to as“tauopa- thies.”Another tauopathy, the rare hereditary disorder known as“frontotemporal dementia with parkinsonism linked to chromosome 17”(FTDP-17), has become a useful proxy for studying tau- related pathology.^65,66One study expressed human wild-type and FTDP-17 mutant tau inC. ele- gans, finding that the FTDP-17 worms had more serious neuronal degeneration and behavioral deficits.⁶⁷Miyasaka et al. conducted a similar experiment where human and mutant tau were selec- tively expressed in mechanosensory neurons.⁶⁸Whereas the worms with wild-type tau experienced a slight decline in the touch response, worms expressing mutant tau displayed a serious and pro- gressive deterioration in sensitivity.

Thus,C. elegansis an elegant model for cellular and elementary systems-level analysis of how aging-related processes influence the nervous system. Because its genome and biology are highly conserved, insights from this organism are likely to be directly relevant to the human system. Fur- thermore, its simplicity and relatively short lifespan can increase its tractability. That being said, another result of its short lifespan is that interpretations of physiological processes—such as oxida- tive stress, caloric restriction, or protein aggregation—are difficult to generalize to humans. There- fore, findings from this organism need to be validated in other model organisms with higher genomic similarity to humans prior to their translation.

Fruit fly

Drosophila melanogaster, the fruit fly, is another important model organism. This system is useful for many reasons, particularly its short generation cycle, low cost, and ease of genetic manipulation.^69,70Aside from accessibility,Drosophilashares a surprising amount of biologically significant pathways with humans, facilitating many breakthroughs in development, sensory pro- cessing, aging, and oxidative stress.^71,72In total,Drosophilashares 50% of its genes and 75% of its gene transcripts with humans.⁷²

Drosophilapowerfully models the genetics of aging. In flies, lifespan is heritable and seems to be related to the reproductive system.⁷³Two fruit fly strains with different reproductive patterns were compared: one short-lived line that gave birth relatively early in life and a long-lived line that gave birth relatively late.⁷⁴When the germ line was eliminated in the short-lived strain, the difference in lifespan between the two lines was equalized. The existence of a single-gene mutation with the ability to extend lifespan inDrosophilasuggests that similar systems may be conserved in other organisms. Another lifespan-related line is themethuselahmutant. It exhibits a partial loss of func- tion that results in a 35% increase in lifespan and heightened resistance to stress.⁷⁵The gene seems to code for a G-protein coupled receptor, indicating that protein-signaling pathways are intimately related to the aging process.^76,77Other studies have looked into more specific changes that accom- pany aging. Gene expression was found to selectively alter the presence of certain proteins in the antenna and the muscle⁷⁸of fruit flies.^78,79

66 Eric B. Emmons, Youngcho Kim, and Nandakumar S. Narayanan

Toxin accumulation models and oxidative stress models of aging have been another major direc- tion of research in the Drosophilamodel. A correlation exists between longevity and increased resistance to oxidative stress in a particularly long-lived fly strain.⁸⁰In another fly model, lifespan was extended by transgenic expression of heat-shock protein 70, likely due to its reduction of stress-related damage.⁸¹Conversely, an 80% decrease inDrosophilalifespan was induced by elim- inating Cu/Zn superoxide dismutase (CuZnSOD), an enzyme that breaks down oxygen radi- cals.⁸² Studies that augmented CuZnSOD found an increase in longevity in Drosophila.^83,84 However, the evidence has been equivocal. Some studies have failed to see a consistent extension in lifespan due to alteration of oxidation enzymes. Orr and Sohal argue that, though studies in Drosophilahave provided support for oxidative stress theory, they have fallen short of definitively confirming it. In their own work, they have shown that CuZnSOD can influence lifespan in at-risk populations while having almost no effect in wild-type flies.⁸³An interesting corollary to this find- ing is that social interaction with younger or longer-lived flies can increase longevity in CuZnSOD mutants.⁸⁵It has also been demonstrated that enhancement of xenobiotic metabolism, specifically mutation of Keap1 and subsequent activation of Nrf2, can increase lifespan.^86,87

Drosophilaresearch has provided particular insight and perspective on PD, especially in terms of cellular and molecular advances. For example, Feany and Bender created a PD model inDrosophila through inclusion of humanα-synuclein protein.⁸⁸By expressing normal and mutantα-synuclein, they found that the mutant protein was associated with locomotor dysfunction, Lewy-body-like protein aggregates, and preferential dopaminergic cell depletion. The progressive motor deficits in the model derive from dysfunction of a subset of dopaminergic neurons.⁸⁹As inC. elegans, LRRK2 is a prominent direction for PD work inDrosophila. Expression of LRRK2 protein inDro- sophila disrupted locomotion, reduced function of dopaminergic neurons, and produced early mortality.⁹⁰However, it has been difficult to determine how important LRRK2 really is, since knockout of the LRRK2 gene has produced variable phenotypic effects.^91,92Finally, parkin is a protein that is considered to be a factor in early-onset PD, and may play a role in the sporadic form of the disease as well.⁹³In flies, parkin knockouts display locomotor deficits, male sterility, and shortened lifespan.⁹⁴Accumulated PINK1 stands as a possible mechanism for these deficits in par- kin-deficient flies. Chen and Dorn found that PINK1 on damaged mitochondrial surfaces phos- phorylates Mfn2, which then acts as a receptor to dock parkin at cardiac mitochondria.⁹⁵

Research into the etiology and treatment of AD is an extremely important area of work in the Drosophilamodel. Similar to AD research inC. elegans, expression of proteins such as tau is a major emphasis. Wittmann et al. showed that expression of wild-type or mutant (R406W) tau repro- duced several of the chief deficits in AD: adult-onset neurodegeneration, premature death, and abnormal accumulation of tau protein.⁹⁶Shulman et al. evaluated 67 genes identified in human genome-wide association studies and found that several were modifiers of the tau pathway.⁹⁷ Another leading factor in the development of AD is the previously mentioned accumulation of amyloid-beta (Aβ) peptides. Work inDrosophilahas found that Aβ40 and Aβ42 are both sufficient to produce learning deficits, while Aβ42 also results in the formation of Aβdeposits and neurode- generation.⁹⁸Presenilins are another family of proteins implicated in AD. These proteins have been correlated with the age of onset of model AD in fruit flies, creating one more target for drug treat- ment in theDrosophilamodel.⁹⁹Drosophilamodels of tau overexpression also have progressive neurodegeneration, possibly linked with chromatin instability.¹⁰⁰Importantly, this work indicates that tau overexpression can interact with other aging-related mechanisms, such as oxidative stress.¹⁰¹

Rodent

The next model organisms that we will address are the primary rodent systems, the mouse (Mus musculus) and the rat (Rattus norvegicus). As mammals, rodents allow more direct comparison to humans than is possible with the worm and fly models. Furthermore, their short generation cycle 67 Animal Models of Pathological Aging

facilitates cellular and genetic manipulations. Genetic alterations in mice are necessarily much less dramatic than those inC. elegansorDrosophilabut can be extremely informative. For example, the IGF-1 (insulin-like growth factor 1) pathway has been implicated in lifespan extension from nema- todes to humans, including mice.¹⁰²However, studies linking this pathway to aging in mice and humans have done so in a more modest, correlative way.^103,104

A variety of other genetic pathways have been investigated for their role in aging. The pathway of protein p66^shchas provided one such model of extended lifespan in mice. This protein appears to be involved in cell apoptosis, with its knockout resulting in heightened resistance to stress and increased longevity.¹⁰⁵Another genetic alteration resulting in increased lifespan is a deletion of S6K1 (ribosomal S6 protein kinase 1), a protein involved in the mTOR (mammalian target of rapa- mycin) pathway. Selman et al. found that knockout of S6K1 mimicked the longevity-enhancing effects of caloric restriction.¹⁰⁶A more recent study found that direct administration of rapamycin, an antagonist for this pathway, was able to increase lifespan in both female and male rats.¹⁰⁷

A different genetic approach lies in the modification of telomere length. When telomeres were extended in mice with increased cancer resistance, researchers were able to delay aging in mice.¹⁰⁸ A complementary approach to studying extended aging is the use of accelerated disorders of aging such as Hutchinson-Gilford progeria syndrome. In a mouse model of HGPS, Osorio et al. knocked in a mutant form of the lamin A/C (LMNA) gene (in human). This mutation resulted in a close representation of human HGPS, especially in terms of the production of the malformed protein progerin.²³

The oxidative stress theory of aging has been studied fairly extensively in mice. However, the results conflict with the robust findings inC. elegansandDrosophila. Altering components of oxi- dative stress pathways has not produced clear changes in the lifespan of rodents.¹⁶Ames dwarf mice constitute one example of lifespan extension that may be related to oxidative stress. These mice exhibit dwarfism, live 50% longer, and lack several hormones, including growth hormone.¹⁰⁹They also have greater levels of the previously mentioned CuZnSOD and lower levels of ROS.¹¹⁰This pattern is complicated by the fact that short-lived mice overexpressing growth hormone also have higher levels of CuZnSOD.¹¹¹The Snell and Laron mouse strains exhibit similar phenotypes to Ames dwarf mice that seem to be associated with mutations in the IGF-1 pathway.¹¹²The muta- tion in the p66^shcprotein pathway discussed above is associated with increased oxidative resistance, both on the organismal and cellular levels.¹¹³In order to elucidate the significance of oxidative stress in aging, Pérez et al. summarized eight years of research into the causal role of various anti- oxidants. Following the genetic manipulation of 18 different antioxidants, they found that only mice null for theSod1gene exhibited an altered phenotype.¹⁶Given the fact that knockout and transgenic mice displayed little change in viability, the authors concluded that oxidative stress might not play a very significant role in the molecular basis of mammalian aging.

Rodents are the most prevalent systems in which to study PD. They have essentially all of the structures of the human brain but with fewer neurons, and are much easier to work with than pri- mates. Additionally, the mouse genome has been sequenced and features a suite of transgenic tools. Furthermore, these manipulations are more generalizable to humans than those in inverte- brate organisms like C. elegans and Drosophila. We begin with transgenic mouse models for α-synuclein. Though they all exhibit some of the hallmarks of Parkinson’s pathology, they fail to capture the progressive midbrain neuronal loss leading to motor and cognitive symptoms.¹¹⁴ α-synuclein mouse models are good representations of most aspects of PD pathology, producing abnormal cell loss.¹¹⁵The A53Tα-synuclein transgenic model is noteworthy as it presents the entire range of pathology associated with humanα-synuclein.⁶⁶Unfortunately, mice transgenic for LRRK2 have failed to display many deficits.⁶³In a similar vein, parkin knockout mice don’t exhibit behavioral abnormalities or dopaminergic dysfunction.¹¹⁶The lack of substantial defect in genetic mouse models differs with genetic human evidence, leading Dawson et al. to posit some genetic resistance mechanism in mice.⁶³Notably, most PD cases are sporadic rather than genetic, developing over several years, implicating some gene-environment interaction.

68 Eric B. Emmons, Youngcho Kim, and Nandakumar S. Narayanan

In contrast, toxin-based models accurately model the dopamine dysfunction observed in PD.

The 6-Hydroxydopamine (6-OHDA) rat model is well known for the fairly selective degeneration of dopaminergic neurons in the substantia nigra (SN) that result in pronounced motor defi- cits.112,117,118,119

Administration of the herbicide paraquat provides a decent model of the Parkin- son’s phenotype. In mice, treatment leads to dopaminergic degeneration in the SN and α-synuclein aggregation.^120,121 MPTP is another well-described toxin model of PD, resulting in the accumulation of α-synuclein and apoptosis of dopaminergic cells in the SN.122,123,124

Despite the impressive range of rodent PD models, no single approach captures all of the significant aspects of the disorder. Though this should qualify subsequent conclusions, it doesn’t limit their promise and should not deter their use.

AD research relies heavily on rodents, especially transgenic mouse models. Most rodent models of Alzheimer’s attempt to replicate the disease by overexpressing Aβand/or tau proteins. Gener- ally, this is done by transgenic induction of amyloid precursor proteins (APPs) or expression of human or mutant tau. As two of the hallmarks of the disorder, it is notable that AD featuring exclu- sively Aβand tau pathologies is uncommon. Rather than being“pure”Alzheimer’s, the disease usually co-occurs with other defects like infarcts.¹²⁵The presentation of the disease is similar in both the sporadic and familial early-onset forms of the disease.¹²⁶Because of this similarity, trans- genic approaches that manipulate Aβand tau are relevant to sporadic cases as well. Although auto- somal-dominant APP mutations are able to induce full-blown AD in humans, they do not in transgenic mice, failing to produce tau-based NFTs.²⁰However, the expression of human tau does generate NFTs.²⁰Over 20 APP mutations have been identified, giving a wide range of targets for genetic models. Strategies that work to manipulate tau must be considered carefully: though involved in frontotemporal dementia (FTD), no known form of heritable AD involves tau muta- tions.²⁰Despite this caveat, the combination of mutations in APP and tau has produced a number of striking models of AD.¹²⁷These studies have been fruitful, indicating that APPs may be involved in the initiation of AD pathology while tau proteins are implicated in its mediation.²⁰In mice, mutant tau overexpression can contribute to neurodegeneration, while suppressing tau can protect against neurodegeneration and improve memory function.^128,129While it is clear that mutant and overexpressed tau both contribute to neuronal dysfunction, the exact mechanisms in mammalian systems remain unclear.¹³⁰Given the failures of recent clinical trials targeting tau accumulation, it is of paramount importance to identify these mechanisms in order to develop new treatments.

Despite the fact that none of these systems are perfect analogues of Alzheimer’s in humans, they can provide valuable insight into risk factors and disease mechanisms.

Rodents are also well suited to model human age-related hearing loss. They have similar sensory- neural transduction, brainstem nuclei, and lower-level auditory processing to humans.¹³¹In addi- tion, rodents rely on hearing and live long enough to experience age-related hearing loss. These homologies and the added power of transgenic models mean that rodent hearing can be evaluated at the cellular, structural, and behavioral levels. These strengths are tempered by the fact that mice hear a very different range of frequencies than humans.¹³²Mice with abnormal growth factors, endolymph, sensory transduction machinery, extracellular matrix, or potassium channels can have profoundly disrupted hearing. In aging, a key vulnerability is the hair cell, which is uniquely sus- ceptible to dysfunction and exposure-related hearing loss. Dysfunction in afferent neurons, sensory neurons, and even the balance of proteins exchanged between them can contribute to aging- related deficits.¹³³As there is no biochemical marker for age-related hearing loss, mouse models of aging tend to model structural, molecular, or behavioral patterns of hearing loss. For instance, mice with mutations in the allele forCdh²³have abnormalities in hearing organs and develop fre- quency-dependent hearing loss.¹³⁴Other processes can influence age-related hearing loss. For instance, mutations in CuZnSOD can influence hearing.¹³⁵Evidence from mice as well as from other model organisms suggests an interaction between noise-related hearing loss and age. In mice, early exposure to high levels of noise appears to predispose animals to hearing loss later in life.^136,137More details on this subject can be found in chapter 16.

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