- Circadian clocks coordinate molecular, cellular, physiological, and behavioral processes with the 24-hour solar day. While clock functions are well understood in young animals, it is not clear how aging or neurodegenerative disease affects the various levels of the circadian system. A common symptom of many neurodegenerative diseases including Alzheimer’s disease (AD) is severe disruptions in sleep/wake cycles, which often begin before the onset of clinical diagnosis. However, it is not known whether disruptions in the circadian clock contribute to AD pathology or whether AD disrupts the circadian clock machinery. To address these questions, we measured lifespan and neurodegeneration in three different Drosophila AD models with and without functioning clocks. Lifespan and neurodegenerative phenotypes correlated with the pathogenicity of the AD models used; however, the loss of circadian clock function did not exacerbate these phenotypes. We also determined that the expression of amyloidogenic peptides disrupt locomotor activity rhythms, climbing ability, and lifespan; however, PER protein oscillation remained rhythmic in the pacemaker neurons, which constitute central clock responsible for behavioral rhythms. These results suggest that AD-
induced sleep/activity rhythm disruption is not due to the loss of central pacemaker neurons, but may disrupt downstream output pathways.
In addition to the central clock, many cells/tissues in Drosophila are known to possess circadian oscillators, including sensory neurons and glia of the central nervous system. Glial cells are divided into several subtypes that have distinct morphological and functional differences, and it is not known which of them contain clock mechanism. To address this question, we took advantage of the glia subtype specific GAL4 drivers to express GFP specifically in perineurial, subperineurial, cortex, and ensheathing glia, as well as astrocytes. Samples were prepared at two-time points corresponding to the peak and trough of PER accumulation in pacemaker neurons. Using immunofluorescence, we determined that perineurial, subperineurial, cortex and ensheathing glia express PER protein in a manner suggestive of circadian oscillation. On the other hand, PER signal was not detected in astrocytes of the central brain at either time point. Since total PER protein in the heads of Drosophila decline with age, we asked whether the intensity of PER signal is altered with age in each of the PER positive glial subtypes. We quantified PER signal intensity in the PER positive glia subtypes in the brains of young and old flies and found that PER levels decreased significantly in perineurial, subperineurial, and cortex glia of old flies. The intensity of PER staining in ensheathing glial cells was highly variable with no significant difference between age groups, and no PER was detected in astrocytes. These results demonstrate that most glia subtypes of Drosophila express PER protein in a manner suggestive of circadian oscillation and that the nighttime accumulation of PER is reduced in surface and cortex glia with age.
Aging is also accompanied by changes in the expression of clock-controlled genes. A recent report demonstrated highly increased expression of a subset of stress-responsive and metabolic genes in the heads of old flies raising the question of whether or not this increase is neuroprotective. To address this question, we focused on lactate dehydrogenase (Ldh), which displayed the highest mRNA increase
in old flies. Ldh encodes the metabolic enzyme LDH that catalyzes the interconversion of pyruvate and lactate with concurrent changes in the NAD+/NADH redox pair. We determined that the heads of old flies contain higher levels of LDH protein, increased enzymatic activity elevated lactate levels. To determine whether increased Ldh expression is beneficial to detrimental to aging flies, we assessed longevity in flies with adult specific overexpression or reduction of Ldh. Adult specific Ldh overexpression in clock-containing cells, neuron, or glia significantly reduced fly lifespan while RNAi mediated Ldh knockdown resulted in lifespan extension. We also determined that neuronal overexpression of Ldh caused significant loss of rest/activity rhythms and increased levels neurodegeneration while reducing Ldh expression in neurons alone resulted in delayed neurodegeneration. These data suggest that age-related increase in Ldh expression negatively affects longevity, neuronal health, and sleep/activity rhythms.
Taken together, research conducted for this dissertation characterized multiple connections between age-related changes in circadian function and metabolism on the healthspan and lifespan of Drosophila melanogaster. Because most molecular pathways involved in systems that we investigated are conserved, we hope that this research on a model organism may contribute to our understanding of the much more complicated connections between aging, circadian system, and neurodegenerative diseases in humans.