Jeremy M. Van Raamsdonk

Deletion of the Mitochondrial Superoxide Dismutase sod-2 Extends Lifespan in Caenorhabditis elegans

The oxidative stress theory of aging postulates that aging results from the accumulation of molecular damage caused by reactive oxygen species (ROS) generated during normal metabolism. Superoxide dismutases (SODs) counteract this process by detoxifying superoxide. It has previously been shown that elimination of either cytoplasmic or mitochondrial SOD in yeast, flies, and mice results in decreased lifespan. In this experiment, we examine the effect of eliminating each of the five individual sod genes present in Caenorhabditis elegans. In contrast to what is observed in other model organisms, none of the sod deletion mutants shows decreased lifespan compared to wild-type worms, despite a clear increase in sensitivity to paraquat- and juglone-induced oxidative stress. In fact, even mutants lacking combinations of two or three sod genes survive at least as long as wild-type worms. Examination of gene expression in these mutants reveals mild compensatory up-regulation of other sod genes. Interestingly, we find that sod-2 mutants are long-lived despite a significant increase in oxidatively damaged proteins. Testing the effect of sod-2 deletion on known pathways of lifespan extension reveals a clear interaction with genes that affect mitochondrial function: sod-2 deletion markedly increases lifespan in clk-1 worms while clearly decreasing the lifespan of isp-1 worms. Combined with the mitochondrial localization of SOD-2 and the fact that sod-2 mutant worms exhibit phenotypes that are characteristic of long-lived mitochondrial mutants—including slow development, low brood size, and slow defecation—this suggests that deletion of sod-2 extends lifespan through a similar mechanism. This conclusion is supported by our demonstration of decreased oxygen consumption in sod-2 mutant worms. Overall, we show that increased oxidative stress caused by deletion of sod genes does not result in decreased lifespan in C. elegans and that deletion of sod-2 extends worm lifespan by altering mitochondrial function.

Caspase Cleavage of Mutant Huntingtin Precedes Neurodegeneration in Huntington's Disease

Huntingtons disease (HD) results from polyglutamine expansion in huntingtin (htt), a protein with several consensus caspase cleavage sites. Despite the identification of htt fragments in the brain, it has not been shown conclusively that htt is cleaved by caspases in vivo. Furthermore, no study has addressed when htt cleavage occurs with respect to the onset of neurodegeneration. Using antibodies that detect only caspase-cleaved htt, we demonstrate that htt is cleaved in vivo specifically at the caspase consensus site at amino acid 552. We detect caspase-cleaved htt in control human brain as well as in HD brains with early grade neuropathology, including one homozygote. Cleaved htt is also seen in wild-type and HD transgenic mouse brains before the onset of neurodegeneration. These results suggest that caspase cleavage of htt may be a normal physiological event. However, in HD, cleavage of mutant htt would release N-terminal fragments with the potential for increased toxicity and accumulation caused by the presence of the expanded polyglutamine tract. Furthermore, htt fragments were detected most abundantly in cortical projection neurons, suggesting that accumulation of expanded htt fragments in these neurons may lead to corticostriatal dysfunction as an early event in the pathogenesis of HD.

Cognitive dysfunction precedes neuropathology and motor abnormalities in the YAC128 mouse model of Huntington's disease.

Huntingtons disease (HD) is an adult-onset neurodegenerative disorder involving motor dysfunction, cognitive deficits, and psychiatric disturbances that result from underlying striatal and cortical dysfunction and neuropathology. The YAC128 mouse model of HD reproduces both the motor deficits and selective degeneration observed in the human disease. However, the presence of cognitive impairment in this model has not been determined. Here, we report mild cognitive deficits in YAC128 mice that precede motor onset and progressively worsen with age. Rotarod testing revealed a motor learning deficit at 2 months of age that progresses such that by 12 months of age, untrained YAC128 mice are unable to learn the rotarod task. Additional support for cognitive dysfunction is evident in a simple swimming test in which YAC128 mice take longer to find the platform than wild-type (WT) controls beginning at 8 months of age. YAC128 mice also have deficits in open-field habituation and in a swimming T-maze test at this age. Strikingly, in the reversal phase of the swimming T-maze test, YAC128 mice take twice as long as WT mice to locate the platform, indicating a difficulty in changing strategy. At 12 months of age, YAC128 mice show decreased prepulse inhibition and habituation to acoustic startle. The clear pattern of cognitive dysfunction in YAC128 mice is similar to the symptoms and progression of cognitive deficits in human HD and provides both the opportunity to examine the relationship between cognitive dysfunction, motor impairment, and neuropathology in HD and to assess whether potential therapies for HD can restore cognitive function.

Superoxide dismutase is dispensable for normal animal lifespan

Reactive oxygen species (ROS) are toxic oxygen-containing molecules that can damage multiple components of the cell and have been proposed to be the primary cause of aging. The antioxidant enzyme superoxide dismutase (SOD) is the only eukaryotic enzyme capable of detoxifying superoxide, one type of ROS. The fact that SOD is present in all aerobic organisms raises the question as to whether SOD is absolutely required for animal life and whether the loss of SOD activity will result in decreased lifespan. Here we use the genetic model organism Caenorhabditis elegans to generate an animal that completely lacks SOD activity (sod-12345 worms). We show that sod-12345 worms are viable and exhibit a normal lifespan, despite markedly increased sensitivity to multiple stresses. This is in stark contrast to what is observed in other genetic model organisms where the loss of a single sod gene can result in severely decreased survival. Investigating the mechanism underlying the normal lifespan of sod-12345 worms reveals that their longevity results from a balance between the prosurvival signaling and the toxicity of superoxide. Overall, our results demonstrate that SOD activity is dispensable for normal animal lifespan but is required to survive acute stresses. Moreover, our findings indicate that maintaining normal stress resistance is not crucial to the rate of aging.

Wild‐type huntingtin protects neurons from excitotoxicity

Huntingtin is a caspase substrate, and loss of normal huntingtin function resulting from caspase‐mediated proteolysis may play a role in the pathogenesis of Huntington disease. Here we tested the hypothesis that increasing huntingtin levels protect striatal neurons from NMDA receptor‐mediated excitotoxicity. Cultured striatal neurons from yeast artificial chromosome (YAC)18 transgenic mice over‐expressing full‐length wild‐type huntingtin were dramatically protected from apoptosis and caspase‐3 activation compared with cultured striatal neurons from non‐transgenic FVB/N littermates and YAC72 mice expressing mutant human huntingtin. NMDA receptor activation induced by intrastriatal injection of quinolinic acid initiated a form of apoptotic neurodegeneration within the striatum of mice that was associated with caspase‐3 cleavage of huntingtin in neurons and astrocytes, decreased levels of full‐length huntingtin, and the generation of a specific N‐terminal caspase cleavage product of huntingtin. In vivo, over‐expression of wild‐type huntingtin in YAC18 transgenic mice conferred significant protection against NMDA receptor‐mediated apoptotic neurodegeneration. These data provide in vitro and in vivo evidence that huntingtin may regulate the balance between neuronal survival and death following acute excitotoxic stress, and that the levels of huntingtin may modulate neuronal sensitivity to excitotoxic neurodegeneration. We suggest that further study of huntingtins anti‐apoptotic function will contribute to our understanding of the pathogenesis of Huntingdons disease and provide insights into the selective vulnerability of striatal neurons to excitotoxic cell death.

Reactive Oxygen Species and Aging in Caenorhabditis elegans: Causal or Casual Relationship?

The free radical theory of aging proposes a causal relationship between reactive oxygen species (ROS) and aging. While it is clear that oxidative damage increases with age, its role in the aging process is uncertain. Testing the free radical theory of aging requires experimentally manipulating ROS production or detoxification and examining the resulting effects on lifespan. In this review, we examine the relationship between ROS and aging in the genetic model organism Caenorhabditis elegans, summarizing experiments using long-lived mutants, mutants with altered mitochondrial function, mutants with decreased antioxidant defenses, worms treated with antioxidant compounds, and worms exposed to different environmental conditions. While there is frequently a negative correlation between oxidative damage and lifespan, there are many examples in which they are uncoupled. Neither is resistance to oxidative stress sufficient for a long life nor are all long-lived mutants more resistant to oxidative stress. Similarly, sensitivity to oxidative stress does not necessarily shorten lifespan and is in fact compatible with long life. Overall, the data in C. elegans indicate that oxidative damage can be dissociated from aging in experimental situations.

Cystamine treatment is neuroprotective in the YAC128 mouse model of Huntington disease

Huntington disease (HD) is an adult onset neurodegenerative disorder characterized by selective atrophy and cell loss within the striatum. There is currently no treatment that can prevent the striatal neuropathology. Transglutaminase (TG) activity is increased in HD patients, is associated with cell death, and has been suggested to contribute to striatal neuronal loss in HD. This work assesses the therapeutic potential of cystamine, an inhibitor of TG activity with additional potentially beneficial effects. Specifically, we examine the effect of cystamine on striatal neuronal loss in the YAC128 mouse model of HD. We demonstrate here for the first time that YAC128 mice show a forebrain‐specific increase in TG activity compared with wild‐type (WT) littermates which is decreased by oral delivery of cystamine. Treatment of symptomatic YAC128 mice with cystamine starting at 7 months prevented striatal neuronal loss. Cystamine treatment also ameliorated the striatal volume loss and striatal neuronal atrophy observed in these animals, but was unable to prevent motor dysfunction or the down‐regulation of dopamine and cyclic adenosine monophsophate‐regulated phosphoprotein (DARPP‐32) expression in the striatum. While the exact mechanism responsible for the beneficial effects of cystamine in YAC128 mice is uncertain, our findings suggest that cystamine is neuroprotective and may be beneficial in the treatment of HD.

Huntingtin inhibits caspase-3 activation

Huntingtons disease results from a mutation in the HD gene encoding for the protein huntingtin. The function of huntingtin, although beginning to be elucidated, remains largely unclear. To probe the prosurvival function of huntingtin, we modulate levels of wild‐type huntingtin in a number of cellular and in vivo models. Huntingtin depletion resulted in caspase‐3 activation, and overexpression of huntingtin resulted in caspase‐3 inhibition. Additionally, we demonstrate that huntingtin physically interacts with active caspase‐3. Interestingly, mutant huntingtin binds active caspase‐3 with a lower affinity and lower inhibitory effect on active caspase‐3 than does wild‐type huntingtin. Although reduction of huntingtin levels resulted in caspase‐3 activation in all conditions examined, the cellular response was cell‐type specific. Depletion of huntingtin resulted in either overt cell death, or in increased vulnerability to cell death. These data demonstrate that huntingtin inhibits caspase‐3 activity, suggesting a mechanism whereby caspase‐mediated huntingtin depletion results in a detrimental amplification cascade leading to further caspase‐3 activation, resulting in cell dysfunction and cell death.

Testicular degeneration in Huntington disease

Huntington disease (HD) is an adult onset, neurodegenerative disorder that results from CAG expansion in the HD gene. Recent work has demonstrated testicular degeneration in mouse models of HD and alterations in the hypothalamic-pituitary-gonadal (HPG) axis in HD patients. Here, we show that HD patients have specific testicular pathology with reduced numbers of germ cells and abnormal seminiferous tubule morphology. In the YAC128 mouse model, testicular degeneration develops prior to 12 months of age, but at 12 months, there is no evidence for decreased testosterone levels or loss of GnRH neurons in the hypothalamus. This suggests that testicular pathology results from a direct toxic effect of mutant huntingtin in the testis and is supported by the fact that huntingtin is highly expressed in the affected cell populations in the testis. Understanding the pathogenesis of HD in the testis may reveal common critical pathways which lead to degeneration in both the brain and testis.

Decreased Energy Metabolism Extends Life Span in Caenorhabditis elegans Without Reducing Oxidative Damage

On the basis of the free radical and rate of living theories of aging, it has been proposed that decreased metabolism leads to increased longevity through a decreased production of reactive oxygen species (ROS). In this article, we examine the relationship between mitochondrial energy metabolism and life span by using the Clk mutants in Caenorhabditis elegans. Clk mutants are characterized by slow physiologic rates, delayed development, and increased life span. This phenotype suggests that increased life span may be achieved by decreasing energy expenditure. To test this hypothesis, we identified six novel Clk mutants in a screen for worms that have slow defecation and slow development and that can be maternally rescued. Interestingly, all 11 Clk mutants have increased life span despite the fact that slow physiologic rates were used as the only screening criterion. Although mitochondrial function is decreased in the Clk mutants, ATP levels are normal or increased, suggesting decreased energy utilization. To determine whether the longevity of the Clk mutants results from decreased production of ROS, we examined sensitivity to oxidative stress and oxidative damage. We found no evidence for systematically increased resistance to oxidative stress or decreased oxidative damage in the Clk mutants despite normal or elevated levels of superoxide dismutases. Overall, our findings suggest that decreased energy metabolism can lead to increased life span without decreased production of ROS.