Skeletal muscle atrophy is a common and often debilitating complication of diverse stresses including muscle disuse, fasting, aging, critical illness and many chronic illnesses. However, the pathogenesis of muscle atrophy is still poorly understood. The thesis herein describes my studies investigating the molecular mechanisms of skeletal muscle atrophy. Using mouse skeletal muscle and cultured skeletal myotubes as experimental systems, I discovered a novel stress-induced pathway in skeletal muscle that causes muscle atrophy. The pathway begins with stress-induced expression of ATF4, a basic leucine zipper (bZIP) transcription factor with an evolutionarily ancient role in cellular stress responses. I found that diverse stresses including fasting and muscle disuse increase expression of ATF4 in skeletal muscle. ATF4 then activates the growth arrest and DNA damage-inducible 45a (Gadd45a) gene, leading to increased expression of Gadd45a protein, an essential and inducible subunit of DNA demethylase complexes. Gadd45a localizes to skeletal myonuclei where it interacts with and stimulates demethylation of a specific region in the promoter of the cyclin dependent kinase inhibitor 1a (Cdkn1a) gene. By demethylating the Cdkn1a promoter, Gadd45a activates the Cdkn1a gene, leading to increased expression of Cdkn1a protein, also known as p21WAF1/CIP1. Cdkn1a stimulates protein breakdown (a critical pro-atrophy process) and inhibits anabolic signaling, protein synthesis and PGC-1α expression (processes that maintain healthy skeletal muscle and protect against atrophy). As a result, Cdkn1a causes skeletal muscle fibers to undergo atrophy. Importantly, interventions that reduce any one component of this pathway (ATF4, Gadd45a or Cdkn1a) reduce skeletal muscle atrophy during fasting, muscle disuse, and perhaps other skeletal muscle stresses such as illness and aging. Conversely, forced expression of any one component of this pathway is sufficient to cause skeletal muscle fiber atrophy in the absence of upstream stress. These data suggest the ATF4/Gadd45a/Cdkn1a pathway as a potential therapeutic target. Collectively, my studies demonstrate that the sequential, stress-induced expression of ATF4, Gadd45a and Cdkn1a is a critical process in the pathogenesis of skeletal muscle atrophy. This significantly advances our understanding of how muscle atrophy occurs and it opens up new avenues of investigation into the causes and treatment of muscle atrophy.

Skeletal muscle atrophy is a debilitating condition that commonly occurs as a secondary consequence of many acute and chronic medical conditions, including muscle disuse, heart and renal failure, starvation, cancer, HIV/AIDS, and aging. Though it leads to weakness, falls, and fractures, and reduces independence and quality of life for millions of Americans annually, no effective pharmacologic therapies for muscle atrophy exist. This is largely due to a poor understanding of the pathogenesis of skeletal muscle atrophy at a molecular level. In this thesis, I describe my studies into the molecular pathogenesis of skeletal muscle atrophy. Using mouse models, I showed that the gene encoding the pro-atrophy nuclear protein Gadd45a is regulated by distinct pathways after muscle denervation and fasting, and also identified a novel protein regulating skeletal muscle fiber size. First, we demonstrated that denervation-induced muscle atrophy, unlike atrophy mediated by fasting, does not require the bZIP transcription factor ATF4. However, the lysine deacetylase HDAC4 is sufficient to induce Gadd45a mRNA and necessary for Gadd45a mRNA induction after denervation, but not after fasting. Taken together, these data show that Gadd45a is a central convergence point for muscle atrophy caused by several stimuli, and also demonstrate that distinct pathways mediate Gadd45a induction in different models of skeletal muscle atrophy. Second, we identified spermine oxidase as a critical regulator of muscle fiber size. We observed that spermine oxidase mRNA and spermine oxidase protein were reduced by several distinct causes of muscle atrophy (i.e. immobilization, denervation, fasting, and aging). Furthermore, spermine oxidase overexpression increased muscle fiber size, while spermine oxidase knockdown caused muscle fiber atrophy. Restoring spermine oxidase expression significantly attenuated muscle atrophy after limb immobilization, denervation, and fasting. Finally, we identified p21 as a key upstream regulator of spermine oxidase expression, and spermine oxidase as a required mediator of p21-mediated skeletal muscle fiber atrophy. Collectively, these findings greatly advance our understanding of the molecular pathogenesis of skeletal muscle atrophy. These data demonstrate that Gadd45a is a convergence point for multiple pro-atrophy pathways and identify spermine oxidase as a novel therapeutic target for the treatment of skeletal muscle atrophy. These discoveries suggest several important new areas for future research, and further our understanding of this common, debilitating condition.

Alzheimer’s disease (AD), the most common cause of dementia in the elderly, is a gradually progressive degenerative neurological disorder that is characterized by increasing cognitive impairment, characteristic degenerative pathology and brain atrophy. Studies have shown that the progression of AD pathology in the brain develops in a predictable pattern and the pathological changes that take place in brain begin at the microscopic level long before the first signs of memory loss. Structural Magnetic Resonance Imaging (MRI), which has exceptional soft tissue contrast and detailed resolution, is the best way to noninvasively examine changes which occur early in the course of AD. For this dissertation, our aim is to improve the methods for measuring the atrophy of brain structures in AD, as seen on MRI, and to apply these methods to subjects with cognitive impairment. This study has established a new coordinate template to replace the widely used Montreal Neurological Institute (MNI) template for the atlas-based segmentation procedure. The new template was derived from the same structural image as the one used by the Automated Anatomical Labeling (AAL) procedure. The agreement of the newly developed coordinate template and AAL helps to estimate accurate spatial transformation parameters used in warping the AAL to individual subject images. The new template combines the spatial information of the structural image and the frequency information of MNI template. Based on the same principle, a set of customized templates has been developed. The customized template, associated atlas and customized priors match more closely the aging population than the previous template, so as to improve the atlas-based segmentation of regions of interest in AD assessment. Visual Rating System (VRS) of a single coronal slice (MB slice) in MRI has been another valuable method in the assessment of medial temporal lobe atrophy. An automated procedure has been developed in this study to measure the hippocampal area on the same coronal slice so that the labor of human experts in the VRS assessment of hippocampus will be significantly reduced. Finally the methods and materials (template and atlas) developed in this dissertation were applied to cross-sectional studies of subjects with cognitive impairment. We conducted volumetric analysis on subjects and conclude that the data from the new approaches have higher correlations with clinical data, and therefore can be reliably used as part of an AD assessment tool.

Background: Idiopathic parkinsonism is a neurodegenerative syndrome of unknown cause and includes Parkinson’s disease (PD) and atypical parkinsonian disorders. The atypical parkinsonian disorders are: Multiple system atrophy (MSA), progressive supranuclear palsy (PSP) and corticobasal degeneration (CBD). The incidence rates of these diseases in Sweden are largely unknown. The diagnosis of each disease relies mainly on clinical examination although several imaging and laboratory parameters may show changes. A diagnosis based on clinical examination is especially difficult early in the course of each disease; diagnosis is easier later on when disease-charactersistic signs have evolved and become more prominent. However, even in later stages it is not uncommon that patients are misdiagnosed. PD can be divided into subgroups based on the main clinical symptoms, i. e. tremor dominant, postural instability and gait difficulty (PIGD), and indeterminate. The PIGD subtype has worse prognosis including higher risk of dementia. The aims were to study the incidence of idiopathic parkinsonism and the different specific parkinsonian disorders in the Umeåregion and to investigate the patients early in the course of the disease with brainmagnetic resonance tomography (MRI), external anal sphincterelectromyography (EAS-EMG) and oculomotor examination. Can these methods improve the differential diagnostic work-up and/or differentiate between the subtypes of PD? Methods: We examined all patients in our catchment area (142,000 inhabitants) who were referred to us due to a suspected parkinsonian syndrome. Our clinic is the only clinic in the area receiving referrals regarding movement disorders. During the period (January 1, 2004 through April 30,2009) 190 patients fulfilled the inclusion criteria and were included in the study. Healthy volunteers served as controls.  Results: Incidence: We found the highest incidences reported in the literature: PD (22.5/100,000/year), MSA(2.4/100,000/year), and PSP (1.2/100,000/year). No CBD patients were encountered. Brain MRI: Degenerative changes were common both in controls and PD. There were no differences between the PD subtypes. EAS-EMG: Pathological changes in EAS-EMG examination were common in PD, MSA and PSP. It was not possible to separate PD, MSA and PSP by the EAS-EMG examination. Oculomotor examination: Pathological results were common in all diagnosis groups compared to controls. It was not possible to separate PD, MSA and PSP or the PD subtypes with the help of oculomotor examination. Conclusions: The incidences of idiopathic parkinsonism, PD, MSA and PSP were higher than previously reported in the literature. It is not clear weather this is due to a true higher incidence in the Umeå region or a more effective casefinding than in other studies. MRI, EAS-EMG and oculomotor examination could not contribute to the differential diagnostic work-up between PD, MSA and PSP nor differentiate between PD subtypes early in the course of the disease.

Spinal muscular atrophy is an autosomal recessive genetic disease that results from the loss and/or degeneration of alpha motor neurons in the lower part of the spinal cord. With ~ 1 in 6000 live births per year being affected, this disease is the second leading cause of infant death and is caused by the loss or decrease of the Survival of Motor Neuron protein (SMN). While a lot is known about the role that SMN plays in the cytoplasmic assembly of spliceosomal small nuclear ribonucleoproteins (snRNPs), it remains a crucial question in the field to gain a better understanding of what specific/distinct function(s) SMN might have in motor neurons. We have identified novel interactions between SMN and two RNA-binding proteins (RBPs) known to be components of axonal RNA granules. More specifically, we demonstrated that SMN interacts with HuD and SERBP1 in a direct fashion in foci-like structures along neurites of motor neuron-derived cells. We have also demonstrated that the SMN/HuD interaction is required for the localization of HuD into RNA granules in neurites of motor neuron-derived cells. Furthermore, I have shown that SERBP1 is down-regulated in the absence of normal levels of SMN and, most importantly, that over-expression of SERBP1 can rescue SMA-like neuronal defects using a cell culture model of the disease. These findings may help shed light on the non-canonical molecular pathway(s) involving SMN and RBPs in motor neurons and underscores the possible therapeutic benefits of targeting these RBPs in the treatment of SMA.

Skeletal muscle (SM) atrophy complicates many illnesses, diminishing quality of life and increasing disease morbidity, health resource utilization and health care costs. In animal models of muscle atrophy, loss of SM mass results predominantly from ubiquitin-mediated proteolysis and ubiquitin ligases are the key enzymes that catalyze protein ubiquitination. We have previously shown that ubiquitin ligase Nedd4-1 is up-regulated in a rodent model of denervation-induced SM atrophy and the constitutive expression of Nedd4-1 is sufficient to induce myotube atrophy in vitro, suggesting an important role for Nedd4-1 in the regulation of muscle mass. In this study we generate a Nedd4-1 SM specific-knockout mouse and demonstrate that the loss of Nedd4-1 partially protects SM from denervation-induced atrophy confirming a regulatory role for Nedd4-1 in the maintenance of muscle mass in vivo. Nedd4-1 did not signal downstream through its known substrates Notch-1, MTMR4 or FGFR1, suggesting a novel substrate mediates Nedd4-1’s induction of SM atrophy.

Many researchers have explored the way younger people perceive weight ratios using a variety of methodologies; however, very few researchers have used a more direct ratio estimation procedure, in which participants estimate an actual ratio between two or more weights. Of the few researchers who have used a direct method, the participants who were recruited were invariably younger adults. To date, there has been no research performed to examine how older adults perceive weight-ratios, using direct estimation or any other technique. Past research has provided evidence that older adults have more difficulty than younger adults in perceiving small differences in weight (i.e., the difference threshold for older adults is higher than that of younger adults). Given this result, one might expect that older adults would demonstrate similar impairments in weight ratio estimation compared to younger adults. The current experiment compared the abilities of 17 younger and 17 older adults to estimate weight ratios, using a direct ratio estimation procedure. On any given trial, participants were presented with two weights, and were asked to provide a direct estimate of the ratio, with the heavier in relation to the lighter. The results showed that the participants’ perceived weight ratios increased as a linear function of the actual weight ratios and that compared to younger adults, the older adults overestimated the weight ratios. The age-related overestimation was especially pronounced at higher weight ratios.

<p>Thymus atrophy is highly inducible by stress and prolonged thymus atrophy can contribute to T cell deficiency or inhibit immune recovery after acute peripheral T cell depletion. Little is known regarding the mechanisms driving thymic involution or thymic reconstitution after acute stress. Leptin deficiency in mice results in chronic thymic atrophy, suppressed cell-mediated immunity, and decreased numbers of total lymphocytes, suggesting a role for leptin in regulating thymopoiesis and overall immune homeostasis. Exogenous leptin administration during stress has been shown to protect against thymic damage, yet the mechanisms governing these thymostimulatory effects are currently undefined. Studies herein define the impact of endotoxin-induced thymic damage in the stromal and lymphoid compartment of the thymus and systemic glucocorticoid and cytokine responses in the animal. We report here the novel finding that leptin receptor expression is restricted to medullary thymic epithelial cells in the normal thymus. Using a model of endotoxin-induced acute thymic involution and recovery, we have demonstrated a role for the metabolic hormone leptin in protection of medullary thymic epithelial cells from acute endotoxin-induced damage. We also demonstrated that systemic leptin treatment decreased endotoxin-induced apoptosis of double positive thymocytes and promoted proliferation of double negative thymocytes in vivo through a leptin receptor isoform b-specific mechanism. Leptin treatment increased thymic expression of IL-7, an important soluble thymocyte growth factor produced by medullary thymic epithelial cells. We also found leptin to inhibit systemic glucocorticoid and pro-inflammatory cytokine responses. Using leptin-deficient and leptin receptor-deficient mice in our stress model, we found that endotoxin-induced thymic atrophy was exacerbated in the absence of leptin, despite an inability to mount a proper pro-inflammatory cytokine response. Together, these data support a model in which leptin can function to protect the thymus gland from stress-induced acute damage in part by reduction of systemic corticosteroid and pro-inflammatory cytokine responses, and intrathymically through a mechanism orchestrated by medullary thymic epithelial cells and their soluble mediators (e.g. IL-7). Taken together, these studies suggest a physiological role for leptin signaling in the thymus for maintaining healthy thymic epithelium and promoting thymopoiesis, which is revealed when thymus homeostasis is perturbed by stress.</p> / Dissertation

Skeletal muscle is defined to be atrophic in osteoporosis models and therefore is a potential target tissue for osteoporosis research. The aim of this longitudinal randomized controlled interdisciplinary study was to analyze the functional, histological, ultra-structral and molecular changes and the role of cachectic muscle atrophy inducer TNF-alpha in the skeletal muscles of the ovariectomized (OVX) rat model which mimics postmenopausal osteoporosis. Female Sprague-Dawley rats were randomly assigned to the control, the OVX and the OVX+10&amp / #956 / g/g/week TNF-alpha antagonist (Remicade) treated OVX-TNF groups. Maximum isometric and tetanic-twitch amplitudes were lower than the control group in the OVX group. Maximum isometric twitch amplitudes recovered in the fast-twitch extensor digitorum longus (EDL) muscles but not in the slow-twitch soleus muscles in the OVX-TNF group. The decrease in tetanic-twitch amplitudes recovered in the OVX-TNF group in both muscle types. Splitting and size variations of fibers, central nuclei and well-preserved overall ultrastructure were noted in the OVX and the OVX-TNF groups. Slow-twitch Type I fiber percentage, areas and diameters increased in EDL muscles of the OVX and the OVX-TNF group comparing to the control group. p65 and MyoD immune-labeling increased in OVX group whereas MyoD and C-Rel increased and p50 decreased in OVX-TNF group. Expressions of 61 genes and 42 unidentified transcripts were significantly different between the control, the OVX and the OVX-TNF groups. To sum up TNF-alpha has a role in skeletal muscle dysfunction in OVX rats and TNF-alpha antagonist administration recovered it. But this modulation was not sufficient for total structural recovery.

The sphingolipids are a family of membrane lipids with only a structural role, influencing lipid bilayer properties, but they also act as effector molecules with essential roles in many aspects of cell biology. The sphingolipids ceramide, sphingosine and S1P have shown opposite effects: whereas ceramide and sphingosine usually inhibit proliferation and promote apoptotic responses to different stress stimuli, S1P is known to stimulate cell growth, and promote cell survival. Ceramide can be produced through the de novo synthesis pathway, and by membrane sphingomyelin hydrolysis catalyzed by sphingomyelinases. Both pathways can be activated by the pro-inflammatory cytokine TNFa. Because this cytokine has been shown to promote muscle loss and seems to be crucial in the development of cachexia, we hypothesized that the formation of ceramide, or a metabolite, can be involved in tumor-induced muscle wasting. We investigated the role of ceramide in the in vitro atrophic effects of TNFa on differentiated C2C12 myotubes, by using cell permeant ceramides and inhibitors of sphingolipid metabolism. We observed that TNFa atrophic effects, as evaluated by the reduction in myotube area, are mimicked by exogenous ceramides, supporting the idea that ceramide can participate in muscle atrophy. To verify if ceramide is a mediator of TNFa-induced atrophy, and to identify the metabolites potentially involved, we analyzed the effects of drugs able to block sphingolipid metabolism at different steps: the inhibition of de novo synthesis pathway was unable to restore myotube size in the presence of TNFa whereas the inhibitors of neutral sphingomyelinases reversed TNFa-induced atrophy. Moreover, an accumulation of ceramide and sphingosine induced pro-atrophic effects, whereas sphingosine-1-phosphate had a protective effect. These observations establish that in C2C12 myotubes, ceramide or other downstream metabolites such as sphingosine, produced by the neutral sphingomyelinase pathway in response to TNFa stimulation, participate in cell atrophy. To evaluate the in vivo role of sphingolipids, we treated BalbC mice carrying C26 adenocarcinoma woth Myriocin, an inhibitor of the de novo pathway of ceramide synthesis, that is able to deplete muscle tissue in all sphingolipids, was administered daily to the animals. This treatment partially protected animals against tumor-induced loss of body weight and muscle weight, without affecting the size of tumors. Moreover, myriocin treatment significantly reversed the decrease in myofiber size associated with tumor development, and reduced the expression of atrogenes Foxo3 and Atrogin-1, showing that it was able to protect against muscle atrophy. These results strongly suggest that ceramide, or a downstream sphingolipid metabolite, is involved in tumor-induced muscle atrophy. The sphingolipid pathway thus appears as a new potential target of pharmacological interventions aiming at protecting muscle tissue against atrophy.