This report contains a summation of a variety of articles that have been read and analysed. Each article describes different methods that can be used to detect lesions, optic disks, drusen and exudates in retinal images. I.e. diagnose e.g. Diabetic Retinopathy and Age-Related Macular Degeneration. A general approach is presented, which all methods more or less is based on. Methods to locate the optic disk The PCA  kNN Regression Hough Transform Fuzzy Convergence Vessel Direction Matched Filter Etc. The best method based on result, reliability, number of images and publisher is kNN regression. The result of this method is remarkably good and that brings some doubt about its reliability. Though the method was published at IEEE and that gives the method a more trustful look. A next best method which also is very useful is Vessel Direction Matched Filter. Methods to detect drusen – diagnose Age-Related Macular Degeneration PNN classifier Histogram approach Etc. The best method based on result, reliability, number of images and publisher is the PNN classifier. The method had a sensitivity of 94 % and a specificity of 95 %. 300 images were used in the experiment which was published by the IEEE in 2011. Methods to detect exudates – diagnose Diabetic Retinopathy Morphological techniques Luv colour space, Wiener filter an Canny edge detector. The best method based on result, reliability, number of images and publisher is an experiment called “Feature Extraction”. The method includes the Luv colour space, Wiener filter (remove noise) and the Canny edge detector. / Den här rapporten innehåller en sammanfattning av ett flertal artiklar som har blivit studerade. Varje artikel har beskrivit en metod som kan användas för att upptäcka sjuka förändringar i ögonbottenbilder, det vill säga, åldersförändringar i gula fläcken och diabetisk retinopati. Metoder för att lokalisera blinda fläcken PCA kNN regression Hough omvandling Suddig konvergens Filtrering beroende på kärlens riktning Mm. Den bästa metoden baserat på resultat, pålitlighet, antal bilder och utgivare är kNN regression. De förvånansvärt goda resultaten kan inbringa lite tvivel på huruvida resultaten stämmer. Artikeln publicerades dock av IEEE och det gör artikeln mer trovärdig. Den näst bästa metoden är filtrering beroende på kärlens riktning. Metoder för att diagnosticera åldersförändringar i gula fläcken PNN klassificeraren Histogram Mm. Den bästa metoden baserat på resultat, pålitlighet, antal bilder och utgivare är PNN klassificeraren. Metoden hade en sensitivitet på 94 % och en specificitet på 95 %. 300 bilder användes i experimentet som publicerades av IEEE år 2011. Metoder att diagnosticera diabetisk retinopati Morfologiska tekniker Luv colour space, Wiener filter and Canny edge detector. Den bästa metoden baserat på resultat, pålitlighet, antal bilder och utgivare är ett experimentet som heter ”Feature Extraction”. Experimentet inkluderar Luv colour space, Wiener filter (brus borttagning) och Canny edge detector

Purpose: The main objective of the present study was the investigation of possible influence of lens opacification on macular pigment optical density (MPOD) measurements. Methods: 86 eyes of 64 patients (mean age 73.4(±8.3)years) were included in the study. MPOD was prospectively measured using one-wavelength reflection method (Visucam500, Carl Zeiss Meditec AG) before and after cataract extraction with implantation of a blue-light filtering intraocular lens (AlconSN60WF). The median of the maximum optical density (MaxOD) and the median of the mean optical density (MeanOD) measurements of macular pigment across the subject group were evaluated. Results: Statistically significant differences were noticed between pre-operative and post-operative measurements, the absolute values were generally lower after cataract extraction. The following median(lower/upper quartile) differences across the group were determined: MaxOD -33.8%(-46.2%/-19.1%), MeanOD -44.0%(-54.6%/-26.6%). Larger changes were observed in elderly patients (<70years of age: (n=25eyes) MaxOD -13.4%(-20.5%/3.6%), MeanOD -23.6%(-30.5%/-15.3%) versus patients ≥70years: (n=61eyes) MaxOD -40.5%(-53.2%/-30.1%), MeanOD -47.2%(-57.8%/-40.1%)) and in patients with progressed stage of cataract. MaxOD for lens opacification grade 1:(n=9eyes) -27.4%(-42.1%/-19.6%), 2:(n=26eyes) -35.0%(-44.2%/-25.3%), 3:(n=21eyes) -34.4%(-45.4%/-11.4%), 4:(n=25eyes) -32.6%(-53.2%/-6.4%) and 5:(n=5eyes) -53.5%(-61.7%/-38.7%) and MeanOD for cataract stage 1:(n=9eyes) -42.6%(-46.0%/-26.0%), 2:(n=26eyes) -44.1%(-51.8%/26.2%), 3:(n=21eyes) -45.7%(-54.7%/-24.7%), 4:(n=25eyes) -39.5%(-59.4%/-26.1%), 5:(n=5eyes) -57.0%(-66.1%/-51.4%). Conclusions: As established by comparison of pre- to post-operative measurements, cataract presented a strong effect on MPOD measured by one-wavelength reflection method. Particular care should therefore be taken when evaluating MPOD using this method in elderly patients with progressed stage of cataract. Future optimization of correcting parameters of scattered light and consideration of cataract influence may allow more precise evaluation of MPOD.

Amyotrophic lateral sclerosis (ALS) is a lethal, progressive neurodegenerative disorder that selectively affects both upper and lower motor neurons, leading to muscle weakness, paralysis and death. Despite recent advances in the identification of genes associated with ALS, the quest for a sensitive biomarker for rapid and accurate diagnosis, prognosis, and treatment response monitoring has not been fulfilled. In this thesis, I report a method of quantifying the integrity of motor neurons in vivo using imaging to record uptake and retrograde transport of intramuscularly injected tetanus toxin fragment C (TTC) into spinal motor neurons. This method tracks and profiles progression of disease (transgenic SOD1G93A and PFN1 ALS mice) and detects subclinical perturbations in net transport, as analyzed in C9orf72 transgenic mice. It also defines a progressive reduction in net transport with aging. To address whether our technique enables drug development, I evaluated therapeutic benefits of (1) gene editing and (2) mutant gene silencing (with RNAi targeting SOD1) in SOD1G93A transgenic mice by characterizing their net axonal transport profiles. I constructed a computational model to evaluate key molecular processes affected in net axonal transport in ALS mouse model. The model allows prediction of key parameters affected in a C9ORF72 BAC transgenic mouse line. Prior immunization with tetanus toxoid does not preclude use of this assay, and it can be used repetitively in the same subject. This assay of net axonal transport offers broad clinical application as a diagnostic tool for motor neuron diseases and as a biomarker for rapid detection of benefit from therapies for transport dysfunction in a range of motor neuron diseases.

Glia are the understudied brain cells that perform many functions essential to maintain nervous system homeostasis and protect the brain from injury. If brain damage occurs, glia rapidly adopt the reactive state and elicit a series of cellular and molecular events known as reactive gliosis, the hallmark of many neurodegenerative diseases. However, the molecular pathways that trigger and regulate this process remain poorly defined. The fruit fly Drosophila melanogaster has glial cells that are strikingly similar to mammalian glia, and which also exhibit reactive responses after neuronal injury. By exploiting its powerful genetic toolbox, we are uniquely positioned to identify the genes that activate and execute glial responses to neuronal injury in vivo. In this dissertation, I use Wallerian degeneration in Drosophila as a model to characterize molecular pathways responsible for glia to recognize neural injury, become activated, and ultimately engulf and degrade axonal debris. I demonstrate a novel role for the GEF (guanine nucleotide exchange factors) complex DRK/DOS/SOS upstream of small GTPase Rac1 in glial engulfment activity and show that it acts redundantly with previously discovered Crk/Mbc/dCed-12 to execute glial activation after axotomy. In addition, I discovered an exciting new role for the TNF receptor associated factor 4 (TRAF4) in glial response to axon injury. I find that interfering with TRAF4 and the downstream kinase misshapen (msn) function results in impaired glial activation and engulfment of axonal debris. Unexpectedly, I find that TRAF4 physically associates with engulfment receptor Draper – making TRAF4 only second factor to bind directly to Draper – and show it is essential for Draper-dependent activation of downstream engulfment signaling, including transcriptional activation of engulfment genes via the JNK and STAT transcriptional cascades. All of these pathways are highly conserved from Drosophila to mammals and most are known to be expressed in mouse brain glia, suggesting functional conservation. My work should therefore serve as an excellent starting point for future investigations regarding their roles in glial activation/reactive gliosis in various pathological conditions of the mammalian central nervous system.

A common feature of many neuropathies is axon degeneration. While the reasons for degeneration differ greatly, the process of degeneration itself is similar in most cases. Axon degeneration after axotomy is termed ‘Wallerian degeneration,’ whereby injured axons rapidly fragment and disappear after a short period of latency (Waller, 1850). Wallerian degeneration was thought to be a passive process until the discovery of the Wallerian degeneration slow (Wlds) mouse mutant. In these mice, axons survive and function for weeks after nerve transection. Furthermore, when the full-length protein is inserted into mouse models of disease with an axon degeneration phenotype (such as progressive motor neuronopathy), Wlds is able to delay disease onset (for a review, see Coleman, 2005). Wlds has been cloned and was found to be a fusion event of two neighboring genes: Ube4b, which encodes an ubiquitinating enzyme, and NMNAT-1 (nicotinamide mononucleotide adenylyltransferase-1), which encodes a key factor in NAD (nicotinamide adenine dinucleotide) biosynthesis, joined by a 54 nucleotide linker span (Mack et al., 2001). To address the role of Wlds domains in axon protection and to characterize the subcellular localization of Wlds in neurons, our lab developed a novel method to study Wallerian degeneration in Drosophila in vivo (MacDonald et al., 2006). Using this method, we have discovered that mouse Wlds can also protect Drosophila axons for weeks after acute injury, indicating that the molecular mechanisms of Wallerian degeneration are well conserved between mouse and Drosophila. This observation allows us to use an easily manipulated genetic model to move the Wlds field forward; we can readily identify what Wlds domains give the greatest protection after injury and where in the neuron protection occurs. In chapter two of this thesis, I identify the minimal domains of Wlds that are needed for protection of severed Drosophila axons: the first 16 amino acids of Ube4b fused to Nmnat1. Although Nmnat1 and Wlds are nuclear proteins, we find evidence of a non-nuclear role in axonal protection in that a mitochondrial protein, Nmnat3, protects axons as well as Wlds. In chapter 3, I further explore a role for mitochondria in Wlds-mediated severed axon protection and find the first cell biological changes seen in a Wlds-expressing neuron. The mitochondria of Wlds- and Nmnat3-expressing neurons are more motile before injury. We find this motility is necessary for protection as suppressing the motility with miro heterozygous alleles suppresses Wldsmediated axon protection. We also find that Wlds- and Nmnat3- expressing neurons show a decrease in calcium fluorescent reporter, gCaMP3, signal after axotomy. We propose a model whereby Wlds, through production of NAD in the mitochondria, leads to an increase in calcium buffering capacity, which would decrease the amount of calcium in the cytosol, allowing for more motile mitochondria. In the case of injury, the high calcium signal is buffered more quickly and so cannot signal for the axon to die. Finally, in chapter 4 of my thesis, I identify a gene in an EMS-based forward genetic screen which can suppress Wallerian degeneration. This mutant is a loss of function, which, for the first time, definitively demonstrates that Wallerian degeneration is an active process. The mammalian homologue of the gene encodes a mitochondrial protein, which in light of the rest of the work in this thesis, highlights the importance of mitochondria in neuronal health and disease. In conclusion, the work presented in this thesis highlights a role for mitochondria in both Wlds-mediated axon protection and Wallerian degeneration itself. I identified the first cell biological changes seen in Wlds-expressing neurons and show that at least one of these is necessary for its protection of severed axons. I also helped find the first Wallerian degeneration loss-of-function mutant, showing Wallerian degeneration is an active process, mediated by a molecularly distinct axonal degeneration pathway. The future of the axon degeneration field should focus on the mitochondria as a potential therapeutic target.

The central nervous system (CNS) is composed of two types of cells: neurons that send electrical signals to transmit information throughout the animal and glial cells. Glial cells were long thought to be merely support cells for the neurons; however, recent work has identified many critical roles for these cells during development and in the mature animal. In the CNS, glial cells act as the resident immune cell and they are responsible for the clearance of dead or dying material. After neuronal injury or death, glial cells become reactive, exhibiting dramatic changes in morphology and patterns of gene expression and ultimately engulfing neuronal debris. This rapid clearance of degenerating neuronal material is thought to be crucial for suppression of inflammation and promotion of functional recovery, but molecular pathways mediating these engulfment events remain poorly defined. Drosophila melanogaster is a genetically tractable model system in which to study glial biology. It has been shown that Drosophila glia rapidly respond to axonal injury both morphologically and molecularly and that they ultimately phagocytose the degenerating axonal debris. This glial response to axonal debris requires the engulfment receptor Draper and downstream signaling molecules dCed-6, Shark, and Rac1. However, much remains unknown about the molecular details of this response. In this thesis I show that Drosophila c-Jun kinase (dJNK) signaling is a critical in vivo mediator of glial engulfment activity. In response to axotomy, glial dJNK signals through a cascade involving the upstream MAPKKKs Slipper and TAK1, the MAPKK MKK4, and ultimately the Drosophila AP-1 transcriptional complex composed of JRA and Kayak to initiate glial phagocytosis of degenerating axons. Interestingly, loss of dJNK also blocked injury-induced up-regulation of Draper levels in glia and glial-specific over-expression of Draper was sufficient to rescue phenotypes associated with loss of dJNK signaling. I have identified the dJNK pathway as a novel mediator of glial engulfment activity and show that a primary role for the glial Slipper/Tak1→MKK4→dJNK→dAP-1 signaling cascade is activation of draper expression after axon injury.

Indiana University-Purdue University Indianapolis (IUPUI) / Friedreich’s Ataxia (FRDA) is a rare and poorly understood autosomal recessive disease caused by a pathological deficiency of the mitochondrial protein frataxin. Patients suffer neurodegeneration, ataxia, diabetes, and heart failure. In an effort to understand the mechanisms of heart failure in FRDA, we investigated the role of the protein modification acetylation, which is highly abundant on mitochondrial proteins and has been implicated in regulating intermediary metabolism. Using mouse models of FRDA, we found that cardiac frataxin deficiency causes progressive hyperacetylation of mitochondrial proteins which is correlated with loss of respiratory chain subunits and an altered mitochondrial redox state. Mitochondrial protein hyperacetylation could be reversed by the mitochondria-localized deacetylase SIRT3 in vitro, suggesting a defect in endogenous SIRT3 activity. Consistently, frataxin-deficient cardiac mitochondria showed significantly decreased rates of fatty acid oxidation and complete oxidation to carbon dioxide. However, the degree of protein hyperacetylation in FRDA could not be fully explained by SIRT3 loss. Our data suggested that intermediary metabolites and perhaps acetyl-CoA, which is required for protein acetylation, are accumulating in frataxin-deficient mitochondria. Upon testing the hypothesis that mitochondrial protein acetylation is non-enzymatic, we found that the minimal chemical conditions of the mitochondrial matrix are sufficient to cause widespread non-enzymatic protein acetylation in vitro. These data suggest that mitochondrial protein hyperacetylation in FRDA cardiomyopathy mediates progressive post-translational suppression of mitochondrial oxidative pathways which is caused by a combination of SIRT3 deficiency and, likely, an accumulation of unoxidized acetyl-CoA capable of initiating non-enzymatic protein acetylation. These findings provide novel insight into the mechanisms underlying a poorly understood and fatal cardiomyopathy and highlight a fundamental biochemical mechanism that had been previously overlooked in biological systems.

Indiana University-Purdue University Indianapolis (IUPUI) / Sterol Regulatory Element Binding Protein-1 (SREBP-1) is a conserved transcription factor of the basic helix-loop-helix leucine zipper family (bHLH-Zip) that primarily regulates glycolytic and lipogenic enzymes such as L-pyruvate kinase, acetyl-CoA carboxylase, fatty acid synthase, stearoyl-CoA desaturase 1, and mitochondrial glycerol-3-phosphate acyltransferase 1. SREBP-1c activity is higher in the liver of human obese patients, as well as ob/ob and db/db mouse models of obesity and type 2 diabetes, underscoring the role of this transcription factor as a contributor to hepatic steatosis and insulin resistance. Nonetheless, SREBP-1 deficient ob/ob mice, do not display improved glycemia despite a significant decrease in hepatic lipid accumulation, suggesting that SREBP-1 might play a role at regulating carbohydrate metabolism. By silencing SREBP-1 in the liver of normal and type 2 diabetes db/db mice, we showed that indeed, SREBP-1 is needed for appropriate regulation of glycogen synthesis and gluconeogenesis enzyme gene expression. Depleting SREBP-1 activity more than 90%, resulted in a significant loss of glycogen deposition and increased expression of Pck1 and G6pc. Hence, the benefits of reducing de novo lipogenesis in db/db mice were offset by the negative impact on gluconeogenesis and glycogen synthesis. Some studies had also indicated that SREBP-1 regulates the insulin signaling pathway, through regulation of IRS2 and a subunit of the PI3K complex, p55g. To gain insight on the consequences of silencing SREBP-1 on insulin sensitivity, we analyzed the insulin signaling and mTOR pathways, as both are interconnected through feedback mechanisms. These studies suggest that SREBP-1 regulates S6K1, a downstream effector of mTORC1, and a key molecule to activate the synthesis of protein. Furthermore, these analyses revealed that depletion of SREBP-1 leads to reduced insulin sensitivity. Overall, our data indicates that SREBP-1 regulates pathways important for the fed state, including lipogenesis, glycogen and protein synthesis, while inhibiting gluconeogenesis. Therefore, SREBP-1 coordinates multiple aspects of the anabolic response in response to nutrient abundance. These results are in agreement with emerging studies showing that SREBP-1 regulates a complex network of genes to coordinate metabolic responses needed for cell survival and growth, including fatty acid metabolism; phagocytosis and membrane biosynthesis; insulin signaling; and cell proliferation.

Indiana University-Purdue University Indianapolis (IUPUI) / Huntington’s Disease (HD) is an inherited, autosomal dominant, neurodegenerative disorder. There is no cure for HD and the existing therapies only alleviate HD symptoms without eliminating the cause of this neuropathology. HD is linked to a mutation in the huntingtin gene, which results in an elongation of the poly-glutamine stretch in the huntingtin protein (Htt). A major hypothesis is that mutant Htt (mHtt) leads to aberrant Ca2+ homeostasis in affected neurons. This may be caused by increased Ca2+ influx into the cell via the N-methyl-Daspartate (NMDA)-subtype of glutamate receptors. The contribution of two major Ca2+ removal mechanisms, mitochondria and plasmalemmal Na+/Ca2+ exchangers (NCX), in neuronal injury in HD remains unclear. We investigated Ca2+ uptake capacity in isolated synaptic (neuronal) and nonsynaptic mitochondria from the YAC128 mouse model of HD. We found that both Htt and mHtt bind to brain mitochondria and the amount of mitochondriabound mHtt correlates with increased mitochondrial Ca2+ uptake capacity. Mitochondrial Ca2+ accumulation was not impaired in striatal neurons from YAC128 mice. We also found that expression of the NCX1 isoform is increased with age in striatum from YAC128 mice compared to striatum from wild-type mice. Interestingly, mHtt and Htt bind to the NCX3 isoform but not to NCX1. NCX3 expression remains unchanged. To further investigate Ca2+ homeostasis modulation, we examined the role of collapsin response mediator protein 2 (CRMP2) in wild-type neurons. CRMP2 is viewed as an axon guidance protein, but has been found to be involved in Ca2+ signaling. We found that CRMP2 interacts with NMDA receptors (NMDAR) and disrupting this interaction decreases NMDAR activity. CRMP2 also interacts with and regulates NCX3, resulting in NCX3 internalization and decreased activity. Augmented mitochondrial Ca2+ uptake capacity and an increased expression of NCX1 in the presence of mHtt suggest a compensatory reaction in response to increased Ca2+ influx into the cell. The role of NCX warrants further investigation in HD. The novel interactions of CRMP2 with NMDAR and NCX3 provide additional insight into the complexity of Ca2+ homeostasis regulation in neurons and may also be important in HD neuropathology.

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