Early-adult methionine restriction reduces methionine sulfoxide and extends lifespan in Drosophila.

Methionine restriction (MetR) extends lifespan in various organisms, but its mechanistic understanding remains incomplete. Whether MetR during a specific period of adulthood increases lifespan is not known. In Drosophila, MetR is reported to extend lifespan only when amino acid levels are low. Here, by using an exome-matched holidic medium, we show that decreasing Met levels to 10% extends Drosophila lifespan with or without decreasing total amino acid levels. MetR during the first four weeks of adult life only robustly extends lifespan. MetR in young flies induces the expression of many longevity-related genes, including Methionine sulfoxide reductase A (MsrA), which reduces oxidatively-damaged Met. MsrA induction is foxo-dependent and persists for two weeks after cessation of the MetR diet. Loss of MsrA attenuates lifespan extension by early-adulthood MetR. Our study highlights the age-dependency of the organismal response to specific nutrients and suggests that nutrient restriction during a particular period of life is sufficient for healthspan extension.

GCN2 mediates access to stored amino acids for somatic maintenance during Drosophila ageing.

Many mechanistic theories of ageing argue that a progressive failure of somatic maintenance, the use of energy and resources to prevent and repair damage to the cell, underpins ageing. To sustain somatic maintenance an organism must acquire dozens of essential nutrients from the diet, including essential amino acids (EAAs), which are physiologically limiting for many animals. In Drosophila, adulthood deprivation of each individual EAA yields vastly different lifespan trajectories, and adulthood deprivation of one EAA, phenylalanine (Phe), has no associated lifespan cost; this is despite each EAA being strictly required for growth and reproduction. Moreover, survival under any EAA deprivation depends entirely on the conserved AA sensor GCN2, a component of the integrated stress response (ISR), suggesting that a novel ISR-mediated mechanism sustains lifelong somatic maintenance during EAA deprivation. Here we investigated this mechanism, finding that flies chronically deprived of dietary Phe continue to incorporate Phe into new proteins, and that challenging flies to increase the somatic requirement for Phe shortens lifespan under Phe deprivation. Further, we show that autophagy is required for full lifespan under Phe deprivation, and that activation of the ISR can partially rescue the shortened lifespan of GCN2-nulls under Phe deprivation. We therefore propose a mechanism by which GCN2, via the ISR, activates autophagy during EAA deprivation, breaking down a larvally-acquired store of EAAs to support somatic maintenance. These data refine our understanding of the strategies by which flies sustain lifelong somatic maintenance, which determines length of life in response to changes in the nutritional environment.

Drosophila melanogaster females prioritise dietary sterols for producing viable eggs.

Limiting calories or specific nutrients without malnutrition, otherwise known as dietary restriction (DR), has been shown to extend lifespan and reduce reproduction across a broad range of taxa. Our recent findings in Drosophila melanogaster show that supplementing flies on macronutrient-rich diets with additional cholesterol can extend lifespan to the same extent as DR, while also sustaining high egg production. Thus, DR may be beneficial for lifespan because it reduces egg production which in turn reduces the mother's demand for sterols, thus supporting longer lifespan. It is also possible that mothers live longer and lay more eggs on high sterol diets because the diet triggers enhanced somatic maintenance and promotes egg production, but at the cost of diminished egg quality. To test this, we measured the viability of eggs and development of offspring from mothers fed either cholesterol-sufficient or cholesterol-limiting diets. We found that even when the mother's diet was completely devoid of cholesterol, viable egg production persisted for ∼10 days. Furthermore, we show that sterol-supplemented flies with long lives lay eggs that have high viability and the same developmental potential as those laid by shorter lived mothers on sterol limiting diets. These findings suggest that offspring viability is not a hidden cost of lifespan extension seen in response to dietary sterol supplementation.

Lid wiper epitheliopathy: The influence of multiple lid eversions and exposure time.

PURPOSE: To investigate the effect of multiple lid eversions on lid wiper epitheliopathy (LWE), along with the effect of cumulative lid exposure time and the patterns of associated staining. METHODS: The increase in area of lid wiper staining with lissamine green was compared by everting both the upper eyelids of each subject (i.e. contralateral design), with one eye being everted once for 45 s and the fellow eyelid everted three times, each time for 15 s. This pattern of contralateral eversion was repeated with a total of three eversions in one eye and nine eversions in the fellow eye, with each eye totalling 135 s cumulative exposure to eversion over about 9 min. The LWE area of staining was objectively quantified from slit lamp photography images captured at every lid eversion by 2 masked observers. Two-way repeated measures ANOVAs were used to determine the effect of number of lid eversions and cumulative exposure time on the amount of staining caused. Each image was also categorized into its primary LWE staining pattern, by a masked observer. RESULTS: The multiple eversions condition caused significantly greater LWE than the single eversion condition (p < 0.001), while cumulative exposure time did not have a significant effect on LWE (p = 0.137). Classification of the primary staining patterns revealed that with more eyelid eversions there was a shift from mostly 'no staining' to minor patterns ('short horizontal bands' and 'vertical streaks') and then to more extensive patterns ('broad horizontal bands' and 'comb-shaped'). CONCLUSIONS: The number of eyelid eversions is a confounding factor that should be controlled when investigating LWE, in particular when considering the link with dry eye or contact lens discomfort. However the cumulative exposure time did not appear to influence the LWE magnitude.

Alterations in the Timing of Huperzine A Cerebral Pharmacodynamics in the Acute Traumatic Brain Injury Setting.

Traumatic brain injury (TBI) may affect the pharmacodynamics of centrally acting drugs. Paired-pulse transcranial magnetic stimulation (ppTMS) is a safe and noninvasive measure of cortical gamma-aminobutyric acid (GABA)-mediated cortical inhibition. Huperzine A (HupA) is a naturally occurring acetylcholinesterase inhibitor with newly discovered potent GABA-mediated antiepileptic capacity, which is reliably detected by ppTMS. To test whether TBI alters cerebral HupA pharmacodynamics, we exposed rats to fluid percussion injury (FPI) and tested whether ppTMS metrics of cortical inhibition differ in magnitude and temporal pattern in injured rats. Anesthetized adult rats were exposed to FPI or sham injury. Ninety minutes post-TBI, rats were injected with HupA or saline (0.6 mg/kg, intraperitoneally). TBI resulted in reduced cortical inhibition 90 min after the injury (N = 18) compared to sham (N = 13) controls (p = 0.03). HupA enhanced cortical inhibition after both sham injury (N = 6; p = 0.002) and TBI (N = 6; p = 0.02). The median time to maximum HupA inhibition in sham and TBI groups were 46.4 and 76.5 min, respectively (p = 0.03). This was consistent with a quadratic trend comparison that projects HupA-mediated cortical inhibition to last longer in injured rats (p = 0.007). We show that 1) cortical GABA-mediated inhibition, as measured by ppTMS, decreases acutely post-TBI, 2) HupA restores lost post-TBI GABA-mediated inhibition, and 3) HupA-mediated enhancement of cortical inhibition is delayed post-TBI. The plausible reasons of the latter include 1) low HupA volume of distribution rendering HupA confined in the intravascular compartment, therefore vulnerable to reduced post-TBI cerebral perfusion, and 2) GABAR dysfunction and increased AChE activity post-TBI.

Huperzine A: A promising anticonvulsant, disease modifying, and memory enhancing treatment option in Alzheimer's disease.

Alzheimer's disease (AD) is the most frequent cause of dementia. Besides cognitive deterioration, patients with AD are prone to seizures - more than 20% of patients diagnosed with AD experience at least one unprovoked seizure and up to 7% have recurrent seizures. Although available antiepileptic drugs (AEDs) may suppress seizures in patients with AD, they may also worsen cognitive dysfunction and increase the risk of falls. On the basis of preclinical studies, we hypothesize that Huperzine A (HupA), a safe and potent acetylcholinesterase (AChE) inhibitor with potentially disease-modifying qualities in AD, may have a realistic role as an anticonvulsant in AD.

Decreased astrocytic thrombospondin-1 secretion after chronic ammonia treatment reduces the level of synaptic proteins: in vitro and in vivo studies.

Chronic hepatic encephalopathy (CHE) is a major complication in patients with severe liver disease. Elevated blood and brain ammonia levels have been implicated in its pathogenesis, and astrocytes are the principal neural cells involved in this disorder. Since defective synthesis and release of astrocytic factors have been shown to impair synaptic integrity in other neurological conditions, we examined whether thrombospondin-1 (TSP-1), an astrocytic factor involved in the maintenance of synaptic integrity, is also altered in CHE. Cultured astrocytes were exposed to ammonia (NH4Cl, 0.5-2.5 mM) for 1-10 days, and TSP-1 content was measured in cell extracts and culture media. Astrocytes exposed to ammonia exhibited a reduction in intra- and extracellular TSP-1 levels. Exposure of cultured neurons to conditioned media from ammonia-treated astrocytes showed a decrease in synaptophysin, PSD95, and synaptotagmin levels. Conditioned media from TSP-1 over-expressing astrocytes that were treated with ammonia, when added to cultured neurons, reversed the decline in synaptic proteins. Recombinant TSP-1 similarly reversed the decrease in synaptic proteins. Metformin, an agent known to increase TSP-1 synthesis in other cell types, also reversed the ammonia-induced TSP-1 reduction. Likewise, we found a significant decline in TSP-1 level in cortical astrocytes, as well as a reduction in synaptophysin content in vivo in a rat model of CHE. These findings suggest that TSP-1 may represent an important therapeutic target for CHE. Defective release of astrocytic factors may impair synaptic integrity in chronic hepatic encephalopathy. We found a reduction in the release of the astrocytic matricellular proteins thrombospondin-1 (TSP-1) in ammonia-treated astrocytes; such reduction was associated with a decrease in synaptic proteins caused by conditioned media from ammonia-treated astrocytes. Exposure of neurons to CM from ammonia-treated astrocytes, in which TSP-1 is over-expressed, reversed (by approx 75%) the reduction in synaptic proteins. NF-kB = nuclear factor kappa B; PSD95 = post-synaptic density protein 95; ONS = oxidative/nitrative stress.

Inhibition of NADPH oxidase activation in oligodendrocytes reduces cytotoxicity following trauma.

Spinal cord injury is a debilitating neurological disorder that initiates a cascade of cellular events that result in a period of secondary damage that can last for months after the initial trauma. The ensuing outcome of these prolonged cellular perturbations is the induction of neuronal and glial cell death through excitotoxic mechanisms and subsequent free radical production. We have previously shown that astrocytes can directly induce oligodendrocyte death following trauma, but the mechanisms regulating this process within the oligodendrocyte remain unclear. Here we provide evidence demonstrating that astrocytes directly regulate oligodendrocyte death after trauma by inducing activation of NADPH oxidase within oligodendrocytes. Spinal cord injury resulted in a significant increase in oxidative damage which correlated with elevated expression of the gp91 phox subunit of the NADPH oxidase enzyme. Immunohistochemical analysis confirmed the presence of gp91 phox in oligodendrocytes in vitro and at 1 week following spinal cord injury. Exposure of oligodendrocytes to media from injured astrocytes resulted in an increase in oligodendrocyte NADPH oxidase activity. Inhibition of NADPH oxidase activation was sufficient to attenuate oligodendrocyte death in vitro and at 1 week following spinal cord injury, suggesting that excitotoxicity of oligodendrocytes after trauma is dependent on the intrinsic activation of the NADPH oxidase enzyme. Acute administration of the NADPH oxidase inhibitor apocynin and the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate channel blocker 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione significantly improved locomotor behavior and preserved descending axon fibers following spinal cord injury. These studies lead to a better understanding of oligodendrocyte death after trauma and identify potential therapeutic targets in disorders involving demyelination and oligodendrocyte death.

Amyloid-ß inhibits thrombospondin 1 release from cultured astrocytes: effects on synaptic protein expression.

Among the consequences of Alzheimer disease are disturbances in synaptic integrity that ultimately lead to impaired cognitive functions. Thrombospondins are extracellular matrix proteins that, in the CNS, are predominantly produced by astrocytes and have been implicated in synaptogenesis. This study examined the effects of amyloid-ß (Aß(1-42); Aß) peptide on intracellular and extracellular levels of thrombospondin 1 (TSP-1) in cultured astrocytes. Amyloid-ß caused a significant (1- to 3-fold) increase in astrocytic intracellular levels of TSP-1 (increased retention) that was associated with a reduction of its release from astrocytes. Because Aß is known to induce oxidative stress in astrocytes, we examined the effects of the antioxidants tempol and apocynin on astrocytic TSP-1 levels and release. Treatment of Aß-exposed astrocyte cultures with antioxidants significantly diminished its cellular retention and stimulated its release. Furthermore, the addition of conditioned media derived from Aß-treated cultured astrocytes that contained a reduced TSP-1 content resulted in a significant loss of synaptophysin and PSD95 in cultured neurons. These findings suggest that Aß-mediated reduction in astrocytic TSP-1 release, possibly related to oxidative stress, contributes to the loss of synaptophysin in neurons. Strategies aimed at enhancing the astrocytic release of TSP-1 may have a therapeutic benefit in Alzheimer disease.

Inhibition of astroglial NF-κB enhances oligodendrogenesis following spinal cord injury.

BACKGROUND: Astrocytes are taking the center stage in neurotrauma and neurological diseases as they appear to play a dominant role in the inflammatory processes associated with these conditions. Previously, we reported that inhibiting NF-κB activation in astrocytes, using a transgenic mouse model (GFAP-IκBα-dn mice), results in improved functional recovery, increased white matter preservation and axonal sparing following spinal cord injury (SCI). In the present study, we sought to determine whether this improvement, due to inhibiting NF-κB activation in astrocytes, could be the result of enhanced oligodendrogenesis in our transgenic mice. METHODS: To assess oligodendrogenesis in GFAP-IκBα-dn compared to wild-type (WT) littermate mice following SCI, we used bromodeoxyuridine labeling along with cell-specific immuno-histochemistry, confocal microscopy and quantitative cell counts. To further gain insight into the underlying molecular mechanisms leading to increased white matter, we performed a microarray analysis in naïve and 3 days, 3 and 6 weeks following SCI in GFAP-IκBα-dn and WT littermate mice. RESULTS: Inhibition of astroglial NF-κB in GFAP-IκBα-dn mice resulted in enhanced oligodendrogenesis 6 weeks following SCI and was associated with increased levels of myelin proteolipid protein compared to spinal cord injured WT mice. The microarray data showed a large number of differentially expressed genes involved in inflammatory and immune response between WT and transgenic mice. We did not find any difference in the number of microglia/leukocytes infiltrating the spinal cord but did find differences in their level of expression of toll-like receptor 4. We also found increased expression of the chemokine receptor CXCR4 on oligodendrocyte progenitor cells and mature oligodendrocytes in the transgenic mice. Finally TNF receptor 2 levels were significantly higher in the transgenic mice compared to WT following injury. CONCLUSIONS: These studies suggest that one of the beneficial roles of blocking NF-κB in astrocytes is to promote oligodendrogenesis through alteration of the inflammatory environment.

Reduced extracellular zinc levels facilitate glutamate-mediated oligodendrocyte death after trauma.

Spinal cord injury results in irreversible paralysis, axonal injury, widespread oligodendrocyte death, and white matter damage. Although the mechanisms underlying these phenomena are poorly understood, previous studies from our laboratory indicate that inhibiting activation of the nuclear factor-κB transcription factor in astrocytes reduces white matter damage and improves functional recovery following spinal cord injury. In the current study, we demonstrate that activation of the nuclear factor-κB transcription factor within astrocytes results in a significant increase in oligodendrocyte death following trauma by reducing extracellular zinc levels and inducing glutamate excitotoxicity. By using an ionotropic glutamate receptor antagonist (CNQX), we show that astroglial nuclear factor-κB-mediated oligodendrocyte death is dependent on glutamate signaling despite no change in extracellular glutamate concentrations. Further analysis demonstrated a reduction in levels of extracellular zinc in astrocyte cultures with functional nuclear factor-κB signaling following trauma. Cotreatment of oligodendrocytes with glutamate and zinc showed a significant increase in oligodendrocyte toxicity under low-zinc conditions, suggesting that the presence of zinc at specific concentrations can prevent glutamate excitotoxicity. These studies demonstrate a novel role for zinc in regulating oligodendrocyte excitotoxicity and identify new therapeutic targets to prevent oligodendrocyte cell death in central nervous system trauma and disease.

Nuclear factor-κB activation in Schwann cells regulates regeneration and remyelination.

Schwann cells (SCs) are crucial for peripheral nerve development and regeneration; however, the intrinsic regulatory mechanisms governing postinjury responses are poorly understood. Activation and deacetylation of nuclear factor-κB (NF- κB) in SCs have been implicated as prerequisites for peripheral nerve myelination. Using GFAP-IκBα-dn mice in which NF- κB transcriptional activation is inhibited in SCs, we found no discernable differences in the quantity or structure of myelinated axons in adult facial nerves. Following crush injury, axonal regeneration was impaired at 31 days and significantly enhanced at 65 days in transgenic animals. Compact remyelination and Remak bundle organization were significantly compromised at 31 days and restored by 65 days post injury. Together, these data indicate that inhibition of NF-κB activation in SCs transiently delays axonal regeneration and compact remyelination. Manipulating the temporal activation of nuclear factor-κB in Schwann cells may offer new therapeutic avenues for PNS and CNS regeneration.