Influences of aging and caloric restriction on the transcriptional profile of skeletal muscle from rhesus monkeys

In laboratory rodents, caloric restriction (CR) retards several age-dependent physiological and biochemical changes in skeletal muscle, including increased steady-state levels of oxidative damage to lipids, DNA, and proteins. We have previously used high-density oligonucleotide arrays to show that CR can prevent or delay most of the major age-related transcriptional alterations in the gastrocnemius muscle of C57BL/6 mice. Here we report the effects of aging and adult-onset CR on the gene expression profile of 7,070 genes in the vastus lateralis muscle from rhesus monkeys. Gene expression analysis of aged rhesus monkeys (mean age of 26 years) was compared with that of young animals (mean age of 8 years). Aging resulted in a selective up-regulation of transcripts involved in inflammation and oxidative stress, and a down-regulation of genes involved in mitochondrial electron transport and oxidative phosphorylation. Middle-aged monkeys (mean age of 20 years) subjected to CR since early adulthood (mean age of 11 years) were studied to determine the gene expression profile induced by CR. CR resulted in an up-regulation of cytoskeletal protein-encoding genes, and also a decrease in the expression of genes involved in mitochondrial bioenergetics. Surprisingly, we did not observe any evidence for an inhibitory effect of adult-onset CR on age-related changes in gene expression. These results indicate that the induction of an oxidative stress-induced transcriptional response may be a common feature of aging in skeletal muscle of rodents and primates, but the extent to which CR modifies these responses may be species-specific.

Linking osteopetrosis and pycnodysostosis: Regulation of cathepsin K expression by the microphthalmia transcription factor family

Various genetic conditions produce dysfunctional osteoclasts resulting in osteopetrosis or osteosclerosis. These include human pycnodysostosis, an autosomal recessive syndrome caused by cathepsin K mutation, cathepsin K-deficient mice, and mitf mutant rodent strains. Cathepsin K is a highly expressed cysteine protease in osteoclasts that plays an essential role in the degradation of protein components of bone matrix. Cathepsin K also is expressed in a significant fraction of human breast cancers where it could contribute to tumor invasiveness. Mitf is a member of a helix–loop–helix transcription factor subfamily, which contains the potential dimerization partners TFE3, TFEB, and TFEC. In mice, dominant negative, but not recessive, mutations of mitf, produce osteopetrosis, suggesting a functional requirement for other family members. Mitf also has been found—and TFE3 has been suggested—to modulate age-dependent changes in osteoclast function. This study identifies cathepsin K as a transcriptional target of Mitf and TFE3 via three consensus elements in the cathepsin K promoter. Additionally, cathepsin K mRNA and protein were found to be deficient in mitf mutant osteoclasts, and overexpression of wild-type Mitf dramatically up-regulated expression of endogenous cathepsin K in cultured human osteoclasts. Cathepsin K promoter activity was disrupted by dominant negative, but not recessive, mouse alleles of mitf in a pattern that closely matches their osteopetrotic phenotypes. This relationship between cathepsin K and the Mitf family helps explain the phenotypic overlap of their corresponding deficiencies in pycnodysostosis and osteopetrosis and identifies likely regulators of cathepsin K expression in bone homeostasis and human malignancy.

Lifespan extension and delayed immune and collagen aging in mutant mice with defects in growth hormone production

Single-gene mutations that extend lifespan provide valuable tools for the exploration of the molecular basis for age-related changes in cell and tissue function and for the pathophysiology of age-dependent diseases. We show here that mice homozygous for loss-of-function mutations at the Pit1 (Snell dwarf) locus show a >40% increase in mean and maximal longevity on the relatively long-lived (C3H/HeJ × DW/J)F1 background. Mutant dwJ/dw animals show delays in age-dependent collagen cross-linking and in six age-sensitive indices of immune system status. These findings thus demonstrate that a single gene can control maximum lifespan and the timing of both cellular and extracellular senescence in a mammal. Pituitary transplantation into dwarf mice does not reverse the lifespan effect, suggesting that the effect is not due to lowered prolactin levels. In contrast, homozygosity for the Ghrhrlit mutation, which like the Pit1dw mutation lowers plasma growth hormone levels, does lead to a significant increase in longevity. Male Snell dwarf mice, unlike calorically restricted mice, become obese and exhibit proportionately high leptin levels in old age, showing that their exceptional longevity is not simply due to alterations in adiposity per se. Further studies of the Pit1dw mutant, and the closely related, long-lived Prop-1df (Ames dwarf) mutant, should provide new insights into the hormonal regulation of senescence, longevity, and late life disease.

Lack of the DNA repair protein O6-methylguanine-DNA methyltransferase in histologically normal brain adjacent to primary human brain tumors.

Exposure to exogenous alkylating agents, particularly N-nitroso compounds, has been associated with increased incidence of primary human brain tumors, while intrinsic risk factors are currently unknown. The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is a major defense against the carcinogenicity of N-nitroso compounds and other alkylators. We report here that in 55% (64/117) of cases, histologically normal brain tissue adjacent to primary human brain tumors lacked detectable MGMT activity [methyl excision repair-defective (Mer-) status]. The incidence of Mer- status in normal brain tissue from brain tumor patients was age-dependent, increasing from 21% in children 0.25-19 years of age to 75% in adults over 50. In contrast, Mer- status was found in 12% (5/43) of normal brain specimens from patients operated for conditions other than primary brain tumors and was not age-dependent. The 4.6-fold elevation in incidence of Mer- status in brain tumor patients is highly significant (chi2 = 24; p < or = 0.001). MGMT activity was independent of age in the lymphocytes of brain tumor patients and was present in lymphocytes from six of nine tumor patients whose normal brain specimen was Mer-. DNA polymerase beta, apurinic/apyrimidinic endonuclease, and lactate dehydrogenase activities were present in all specimens tested, including Mer- specimens from brain tumor patients. Our data are consistent with a model of carcinogenesis in human brain in which epigenetically regulated lack of MGMT is a predisposing factor and alkylation-related mutagenesis is a driving force.

Immunohistochemical detection of 4-hydroxynonenal protein adducts in Parkinson disease.

There is growing evidence that oxidative stress and mitochondrial respiratory failure with attendant decrease in energy output are implicated in nigral neuronal death in Parkinson disease (PD). It is not known, however, which cellular elements (neurons or glial cells) are major targets of oxygen-mediated damage. 4-Hydroxy-2-nonenal (HNE) was shown earlier to react with proteins to form stable adducts that can be used as markers of oxidative stress-induced cellular damage. We report here results of immunochemical studies using polyclonal antibodies directed against HNE-protein conjugates to label the site of oxidative damage in control subjects (ages 18-99 years) and seven patients that died of PD (ages 57-78 years). All the nigral melanized neurons in one of the midbrain sections were counted and classified into three groups according to the intensity of immunostaining for HNE-modified proteins--i.e., no staining, weak staining, and intensely positive staining. On average, 58% of nigral neurons were positively stained for HNE-modified proteins in PD; in contrast only 9% of nigral neurons were positive in the control subjects; the difference was statistically significant (Mann-Whitney U test; P < 0.01). In contrast to the substantia nigra, the oculomotor neurons in the same midbrain sections showed no or only weak staining for HNE-modified proteins in both PD and control subjects; young control subjects did not show any immunostaining; however, aged control subjects showed weak staining in the oculomotor nucleus, suggesting age-related accumulation of HNE-modified proteins in the neuron. Our results indicate the presence of oxidative stress within nigral neurons in PD, and this oxidative stress may contribute to nigral cell death.

Serine phosphorylation of human P450c17 increases 17,20-lyase activity: implications for adrenarche and the polycystic ovary syndrome.

Microsomal cytochrome P450c17 catalyzes both steroid 17 alpha-hydroxylase activity and scission of the C17-C20 steroid bond (17,20-lyase) on the same active site. Adrenal 17 alpha-hydroxylase activity is needed to produce cortisol throughout life, but 17,20-lyase activity appears to be controlled independently in a complex, age-dependent pattern. We show that human P450c17 is phosphorylated on serine and threonine residues by a cAMP-dependent protein kinase. Phosphorylation of P450c17 increases 17,20-lyase activity, while dephosphorylation virtually eliminates this activity. Hormonally regulated serine phosphorylation of human P450c17 suggests a possible mechanism for human adrenarche and may be a unifying etiologic link between the hyperandrogenism and insulin resistance that characterize the polycystic ovary syndrome.

A biomarker that identifies senescent human cells in culture and in aging skin in vivo.

Normal somatic cells invariably enter a state of irreversibly arrested growth and altered function after a finite number of divisions. This process, termed replicative senescence, is thought to be a tumor-suppressive mechanism and an underlying cause of aging. There is ample evidence that escape from senescence, or immortality, is important for malignant transformation. By contrast, the role of replicative senescence in organismic aging is controversial. Studies on cells cultured from donors of different ages, genetic backgrounds, or species suggest that senescence occurs in vivo and that organismic lifespan and cell replicative lifespan are under common genetic control. However, senescent cells cannot be distinguished from quiescent or terminally differentiated cells in tissues. Thus, evidence that senescent cells exist and accumulate with age in vivo is lacking. We show that several human cells express a beta-galactosidase, histochemically detectable at pH 6, upon senescence in culture. This marker was expressed by senescent, but not presenescent, fibroblasts and keratinocytes but was absent from quiescent fibroblasts and terminally differentiated keratinocytes. It was also absent from immortal cells but was induced by genetic manipulations that reversed immortality. In skin samples from human donors of different age, there was an age-dependent increase in this marker in dermal fibroblasts and epidermal keratinocytes. This marker provides in situ evidence that senescent cells may exist and accumulate with age in vivo.

Impairment of Intrinsically Photosensitive Retinal Ganglion Cells Associated With Late Stages of Retinal Degeneration

Purpose. To evaluate quantitative and qualitative age-related changes in intrinsically photosensitive melanopsin-containing retinal ganglion cells (ipRGCs) in transgenic P23H rats, an animal model of autosomal dominant retinitis pigmentosa (RP) was examined. Methods. ipRGC density, morphology, and integrity were characterized by immunohistochemistry in retinas extracted from P23H and Sprague–Dawley (SD) rats aged 4, 12, and 18 months. Differences between SD and P23H rats throughout the experimental stages, as well as the interactions among them, were morphologically evaluated. Results. In rat retinas, we have identified ipRGCs with dendrites stratifying in either the outer margin (M1) or inner side (M2) of the inner plexiform layer, and in both the outer and inner plexuses (M3). A small group of M1 cells had their somas located in the inner nuclear layer (M1d). In SD rats, ipRGCs showed no significant changes associated with age, in terms of either mean cell density or the morphologic parameters analyzed. However, the mean density of ipRGCs in P23H rats fell by approximately 67% between 4 and 18 months of age. Moreover, ipRGCs in these animals showed a progressive age-dependent decrease in the dendritic area, the number of branch points and terminal neurite tips per cell, and the Sholl area. Conclusions. In the P23H rat model of retinitis pigmentosa, density, wholeness, and dendritic arborization of melanopsin-containing ganglion cells decrease in advanced stages of the degenerative disease.

Use of space by domestic chicks housed in complex aviaries.

To improve the understanding of the development of locomotor capacity in layer hens, we measured how female laying hen chicks (n=120) of four different strains (LSL-lite, Hyline Brown, Dekalb White, Lohmann Brown; 3 groups of 10 chicks per line) utilized the ground, the air, elevated horizontal (platforms and perches) and inclined surfaces (ramps and ladders) in an aviary until 9 weeks of age. We used infra-red video recordings to perform all-occurrences sampling of locomotive behavioural and perching events that occurred on the ground—where bedding material, food and water were provided, in the air, and on elevated horizontal and inclined surfaces within weekly 30-min sampling periods. Chicks preferred level ground during the first week of life compared to weeks 5–9 (P<0.0001) and performed 52% of all behavioural events in this section. Elevated surface use began at 2 weeks of age and increased over time (P=0.003), where most behaviour was performed in S2 (45% of all events). Chicks preferred horizontal to inclined surfaces, which were used from weeks 2–5 with maximum use occurring during weeks 2 and 3. Lohmann LSL chicks used the space above the ground most frequently (P=0.05) and performed more aerial ascent/descent behaviour than other lines (P<0.0001). Overall activity levels declined with age (P<0.0001). In summary layer chicks almost exclusively locomoted on the ground but utilized elevated horizontal surfaces (perch, first platform) as early as 2 weeks. These results provide information for improving space use in rearing aviaries by introducing lower perches, platforms and ramps/ladders to accommodate age-dependent locomotor abilities.

Mitochondrial impairments contribute to Spinocerebellar ataxia type 1 progression and can be ameliorated by the mitochondria-targeted antioxidant MitoQ

Spinocerebellar ataxia type 1 (SCA1), due to an unstable polyglutamine expansion within the ubiquitously expressed Ataxin-1 protein, leads to the premature degeneration of Purkinje cells (PCs), decreasing motor coordination and causing death within 10-15 years of diagnosis. Currently, there are no therapies available to slow down disease progression. As secondary cellular impairments contributing to SCA1 progression are poorly understood, here, we focused on identifying those processes by performing a PC specific proteome profiling of Sca1154Q/2Q mice at a symptomatic stage. Mass spectrometry analysis revealed prominent alterations in mitochondrial proteins. Immunohistochemical and serial block-face scanning electron microscopy analyses confirmed that PCs underwent age-dependent alterations in mitochondrial morphology. Moreover, colorimetric assays demonstrated impairment of the electron transport chain complexes (ETC) and decrease in ATPase activity. Subsequently, we examined whether the mitochondria-targeted antioxidant MitoQ could restore mitochondrial dysfunction and prevent SCA1-associated pathology in Sca1154Q/2Q mice. MitoQ treatment both presymptomatically and when symptoms were evident ameliorated mitochondrial morphology and restored the activities of the ETC complexes. Notably, MitoQ slowed down the appearance of SCA1-linked neuropathology such as lack of motor coordination as well as preventing oxidative stress-induced DNA / RNA damage and PC loss. Our work identifies a central role for mitochondria in PC degeneration in SCA1 and provides evidence for the supportive use of mitochondria-targeted therapeutics in slowing down disease progression.

Developmental changes in expression of GABA(A) receptor-channels in rat intrinsic cardiac ganglion neurones

The effects of gamma-aminobutyric acid (GABA) on the electrophysiological properties of intracardiac neurones were investigated in the intracardiac ganglion plexus in situ and in dissociated neurones from neonatal, juvenile and adult rat hearts. Focal application of GABA evoked a depolarizing, excitatory response in both intact and dissociated intracardiac ganglion neurones. Under voltage clamp, both GABA and muscimol elicited inward currents at -60 mV in a concentration-dependent manner. The fast, desensitizing currents were mimicked by the GABA(A) receptor agonists muscimol and taurine, and inhibited by the GABA(A) receptor antagonists, bicuculline and picrotoxin. The GABA(A0) antagonist (1,2,5,6-tetrahydropyridin-4-yl)methyl phosphonic acid (TPMPA), had no effect on GABA-induced currents, suggesting that GABA(A) receptor-channels mediate the response. The GABA-evoked current amplitude recorded from dissociated neurones was age dependent whereby the peak current density measured at -100 mV was similar to 20 times higher for intracardiac neurones obtained from neonatal rats (P2-5) compared with adult rats (P45-49). The decrease in GABA sensitivity occurred during the first two postnatal weeks and coincides with maturation of the sympathetic innervation of the rat heart. Immunohistochemical staining using antibodies against GABA demonstrate the presence of GABA in the intracardiac ganglion plexus of the neonatal rat heart. Taken together, these results suggest that GABA and taurine may act as modulators of neurotransmission and cardiac function in the developing mammalian intrinsic cardiac nervous system.

Monitoring Infants By Automatic Video Processing

This work has, as its objective, the development of non-invasive and low-cost systems for monitoring and automatic diagnosing specific neonatal diseases by means of the analysis of suitable video signals. We focus on monitoring infants potentially at risk of diseases characterized by the presence or absence of rhythmic movements of one or more body parts. Seizures and respiratory diseases are specifically considered, but the approach is general. Seizures are defined as sudden neurological and behavioural alterations. They are age-dependent phenomena and the most common sign of central nervous system dysfunction. Neonatal seizures have onset within the 28th day of life in newborns at term and within the 44th week of conceptional age in preterm infants. Their main causes are hypoxic-ischaemic encephalopathy, intracranial haemorrhage, and sepsis. Studies indicate an incidence rate of neonatal seizures of 0.2% live births, 1.1% for preterm neonates, and 1.3% for infants weighing less than 2500 g at birth. Neonatal seizures can be classified into four main categories: clonic, tonic, myoclonic, and subtle. Seizures in newborns have to be promptly and accurately recognized in order to establish timely treatments that could avoid an increase of the underlying brain damage. Respiratory diseases related to the occurrence of apnoea episodes may be caused by cerebrovascular events. Among the wide range of causes of apnoea, besides seizures, a relevant one is Congenital Central Hypoventilation Syndrome (CCHS) \cite{Healy}. With a reported prevalence of 1 in 200,000 live births, CCHS, formerly known as Ondine's curse, is a rare life-threatening disorder characterized by a failure of the automatic control of breathing, caused by mutations in a gene classified as PHOX2B. CCHS manifests itself, in the neonatal period, with episodes of cyanosis or apnoea, especially during quiet sleep. The reported mortality rates range from 8% to 38% of newborn with genetically confirmed CCHS. Nowadays, CCHS is considered a disorder of autonomic regulation, with related risk of sudden infant death syndrome (SIDS). Currently, the standard method of diagnosis, for both diseases, is based on polysomnography, a set of sensors such as ElectroEncephaloGram (EEG) sensors, ElectroMyoGraphy (EMG) sensors, ElectroCardioGraphy (ECG) sensors, elastic belt sensors, pulse-oximeter and nasal flow-meters. This monitoring system is very expensive, time-consuming, moderately invasive and requires particularly skilled medical personnel, not always available in a Neonatal Intensive Care Unit (NICU). Therefore, automatic, real-time and non-invasive monitoring equipments able to reliably recognize these diseases would be of significant value in the NICU. A very appealing monitoring tool to automatically detect neonatal seizures or breathing disorders may be based on acquiring, through a network of sensors, e.g., a set of video cameras, the movements of the newborn's body (e.g., limbs, chest) and properly processing the relevant signals. An automatic multi-sensor system could be used to permanently monitor every patient in the NICU or specific patients at home. Furthermore, a wire-free technique may be more user-friendly and highly desirable when used with infants, in particular with newborns. This work has focused on a reliable method to estimate the periodicity in pathological movements based on the use of the Maximum Likelihood (ML) criterion. In particular, average differential luminance signals from multiple Red, Green and Blue (RGB) cameras or depth-sensor devices are extracted and the presence or absence of a significant periodicity is analysed in order to detect possible pathological conditions. The efficacy of this monitoring system has been measured on the basis of video recordings provided by the Department of Neurosciences of the University of Parma. Concerning clonic seizures, a kinematic analysis was performed to establish a relationship between neonatal seizures and human inborn pattern of quadrupedal locomotion. Moreover, we have decided to realize simulators able to replicate the symptomatic movements characteristic of the diseases under consideration. The reasons is, essentially, the opportunity to have, at any time, a 'subject' on which to test the continuously evolving detection algorithms. Finally, we have developed a smartphone App, called 'Smartphone based contactless epilepsy detector' (SmartCED), able to detect neonatal clonic seizures and warn the user about the occurrence in real-time.

The effect of ageing on in vivo human ciliary muscle morphology and contractility

Purpose. To assess the effect of ageing on in vivo human ciliary muscle morphology and contractility during accommodation. Methods. Seventy-nine subjects, aged 19–70 years were recruited. High-resolution images were acquired of nasal and temporal ciliary muscle in the relaxed state, and at stimulus vergence levels of -4 and -8 D, using anterior segment optical coherence tomography (AS-OCT). Objective refractions and axial lengths were also recorded. Linear regression analysis was performed to determine the effect of age on nasal and temporal ciliary muscle morphologic characteristics. Results. Ciliary muscle anterior length decreased significantly with age both nasally (R = 0.461, P = 0.001) and temporally (R = 0.619, P < 0.001) in emmetropic eyes. In a subset of 37 participants, ciliary muscle maximum width increased significantly with age, by 2.8 µm/year nasally (R = 0.54, P < 0.001) and 3.0 µm/year temporally (R = 0.44, P = 0.007), while the distance from the inner apex of the ciliary muscle to the scleral spur decreased significantly with age on both the nasal and temporal aspects (R = 0.47; P = 0.004 and R = 0.43; P = 0.009, respectively). During accommodation, changes to ciliary muscle thickness and length remained constant throughout life. Conclusions. The human ciliary muscle undergoes age-dependent changes in morphology that suggest an antero-inwards displacement of muscle mass, particularly in emmetropic eyes. However, the morphologic changes observed appear not to affect the ability of the muscle to contract during accommodation, even in established presbyopes, thus supporting a lenticular model of presbyopia development.

The effect of ageing on macrophage Toll-like receptor-mediated responses in the fight against pathogens

The cellular changes during ageing are incompletely understood yet immune system dysfunction is implicated in the age-related decline in health. The acquired immune system shows a functional decline in ability to respond to new pathogens whereas serum levels of cytokines are elevated with age. Despite these age-associated increases in circulating cytokines, the function of aged macrophages is decreased. Pathogen-associated molecular pattern receptors such as Toll-like receptors (TLRs) are vital in the response of macrophages to pathological stimuli. Here we review the evidence for defective TLR signalling in normal ageing. Gene transcription, protein expression and cell surface expression of members of the TLR family of receptors and co-effector molecules do not show a consistent age-dependent change across model systems. However, there is evidence for impaired downstream signalling events, including inhibition of positive and activation of negative modulators of TLR induced signalling events. In this paper we hypothesize that despite a poor inflammatory response via TLR activation, the ineffective clearance of pathogens by macrophages increases the duration of their activation and contributes to perpetuation of inflammatory responses and ageing.

Experiments and observations on the behaviour and brain of the aging female mouse with special reference to dementia of the Alzheimer type

The objective of this research was to investigate the effects of normal aging and the additional effects of chronic exposure to two experimental diets, one enriched in aluminium, the other enriched in lecithin, on aspects of the behaviour and brain histology of the female mouse. The aluminium diet was administered in an attempt to develop a rodent model of Dementia of the Alzheimer Type (DAT). With normal aging, almost all assessed aspects of behaviour were found to be impaired. As regards cognition, selective impairments of single-trial passive avoidance and Morris place learning were observed. While all aspects of open-field behaviour were impaired, the degree of impairment was directly related to the degree of motoric complexity. Deficits were also observed on non-visual sensorimotor coordination tasks and in olfactory discrimination. Histologically, neuron loss, gliosis, vacuolation and congophilic angiopathy were observed in several of the brain regions/fibre tracts believed to contribute to the control of some of the assessed behaviours. The aluminium treatment had very selective effects on both behaviour and brain histology, inducing several features observed in DAT. Behaviourally, the treatment induced impaired spatial reference memory; reduced ambulation; disturbed olfactory function and induced the premature development of the senile pattern of swimming. Histologically, significant neuron loss and gliosis were observed in the hippocampus, entorhinal cortex, amygdala, medial septum, pyriform and pr-frontal cortex. In addition, the brain distribution of congophilic angiopathy was significantly increased by the treatment. The lecithin treatment had effects on both non-cognitive and cognitive aspects of behaviour. The effects of aging on open-field ambulation and rearing were partially ameliorated by the treatment. A similar effect was observed for single-trial passive avoidance performance. Age-dependent improvements in acquisition/retention were observed in 17-23 month mice and Morris place task performance was improved in 11 and 17 month mice. Histologically, a partial sparing of neurons in the cerebellum, hippocampus, entorhinal cortex and subiculum was observed.