Thesis advisor: Thomas N. Seyfried / Thesis advisor: Charles Hoffman / Bis(monoacylglycero)phosphate, BMP, is a negatively charged glycerol-phospholipid with an unusual sn-1;sn-1’ structural configuration. BMP is primarily enriched in endosomal/lysosomal membranes. BMP is thought to play a role in glycosphingolipid degradation and cholesterol transport. It constitutes only about 1-2% of the total phospholipids in most mammalian cells, but is abundant in lung alveolar macrophages where it can comprise up to 16% of the total phospholipids. BMP also accumulates in tissues of humans and animals with lysosomal storage disorders. However, little information is available on BMP levels in gangliosidosis brain tissue. In this work, I found that total BMP content was significantly greater in cells of macrophage/microglial origin than in cells of macroglial origin (astrocyte, oligodendrocyte progenitor), whether normal or tumorigenic. I also observed that BMP in brain was significantly greater in humans and in animals (mice, cats, American black bears) with either GM1 or GM2 ganglioside storage diseases, than in brains of normal subjects. Since BMP is associated with macrophages, I also analyzed the BMP levels in relation to disease-associated inflammation in gangliosidoses. I found that BMP levels were increased due to accumulation of primary storage material gangliosides, rather than an outcome of disease-associated inflammation. In addition, in this thesis I also explored the effect of new ketogenic diet formula from Solace Nutrition (KetoGen) on the growth and metastatic spread of the VM-M3 tumor. Most current drug therapies for cancer are toxic and only marginally effective in providing long-term management. Respiratory insufficiency with compensatory aerobic fermentation (Warburg effect) is the hallmark biochemical phenotype of nearly all neoplastic cells within tumors. Calorie restriction, which lowers blood glucose and elevates ketone bodies, is known to reduce tumor growth to a certain extent, however it does not reduce systemic metastasis. Tumor bearing VM mice were fed either a standard lab chow diet in unrestricted amounts (SD-UR), a standard lab chow restricted to obtain an 18% reduction in body weight (SD-R), or the KetoGen diet restricted (KG-R) to match the body weights of the SD-R group. Tumor size was significantly smaller and organ metastasis was significantly less in the KG-R group than in the SD-UR or SD-R groups. Even though blood glucose was reduced similarly in both the SD-R and KG-R groups, blood ketones were 3-fold higher in the KG-R group than in the SD-R group. These results show that VM-M3 tumor growth and systemic metastasis were managed better with the restricted KetoGen KD than with calorie restriction of a high carbohydrate standard diet. As all human and mouse tumors cells suffer from respiratory insufficiency, my findings suggest that the restricted KetoGen diet should be an effective non-toxic therapy against tumor growth and systemic metastatic cancer. / Thesis (PhD) — Boston College, 2015. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

<p>Glucose metabolism remains an intensely explored topic of cancer biology since the initial discoveries of Otto Warburg nearly 80 years ago. Many solid tumors metabolize glucose primarily to lactate despite the availability of oxygen, revealing a dependence on glycolysis that may serve as a basis for targeted therapy. In particular, a diet devoid of carbohydrate may minimize the growth capabilities of glucose-dependent cancers. As our interests lie in prostate cancer, we examined whether a ketogenic diet devoid of carbohydrate (NCKD) would reduce the growth rate of tumors derived from human prostate cancer cell lines in a murine xenograft model.</p> <p>Our initial experiments utilized the LAPC-4 cell line, a human androgen-sensitive prostate cancer cell line, in a SCID-mouse xenograft model to determine the effects of an NCKD on tumor growth and animal survival relative to two other diets: (1.) a Western-type diet (WD) reflecting consumptions patterns of men diagnosed with prostate-cancer in the Western world and (2.) a low-fat diet (LFD) representing the present standard of care. Following this study, we conducted a second study utilizing a different human prostate cancer cell line (LNCaP) in order to assess whether our initial observations were robust across multiple prostate cancer tumor models and to also further explore the molecular underpinnings of our observations. Both studies revealed the NCKD leads to a reduction in tumor growth rate and greater overall mouse survival relative to the WD. In addition, the NCKD was equivalent in these parameters to the LFD. We also observed key associations between survival and extent of urinary ketosis as well as favorable changes in insulin and insulin-like growth factor-1 (IGF-1) and gene expression that would be predictive of prolonged survival in mice consuming the NCKD.</p> <p>We believe these data provide compelling evidence to consider a potential therapeutic role for dietary carbohydrate restriction in prostate cancer. We hope these results ultimately serve as a basis to conduct future clinical trials assessing whether dietary carbohydrate restriction, either alone or in combination with more conventional therapies, provides clinicians with an additional weapon against prostate cancer.</p> / Dissertation

Dietary manipulations and their pharmacological outcomes have been increasingly studied in neurodegenerative diseases. However, a systematic comparison among different methods in validated animal models of Alzheimer's disease is made necessary due to several different approaches applied in recent studies. Moreover, despite the large body of evidence on the effects of calorie restriction (CR) and ketogenic diets (KDs) on amyloid pathology, no consistent data is available on the effects of calorie restriction, ketogenic diet or ketone supplements on tau pathology in transgenic models of AD. Moreover, the ketogenic diet used in our studies was custom made with low carbohydrate content and rich in medium chain triglyceride (MCT) oils, known to be rapidly metabolized in the liver, resulting in sustained peripheral ketosis. Chapter 1 tested the ability of KD to induce significant ketosis in a mouse model of amyloid deposition. We showed that, despite the mild ketosis induced, KD fed APP mice presented subtle behavioral improvement shown as faster learning in the radial arm water maze, making less errors than APP mice kept on a control diet. Additionally, we observed decreased Aβ immunoreactivity in the anterior cortex of KD fed versus control fed APP mice, despite the lack of changes in congophilic deposits. Due to the mild ketosis induced, a modified ketogenic diet was devised with decreased maltodextrin content and showed greater peripheral levels of β-hydroxybutyrate. Chapter 2 investigated the effects of a ketogenic diet in two transgenic mouse models of Alzheimer's pathology. Interestingly, we found that both transgenic lines, regardless of diet, weighed less than nontransgenic mice, despite their elevated food intake. The reduced body weight may, in part, be explained by the increased locomotor activity shown by both transgenic lines in both the open field and y-maze. Moreover, KD fed mice performed significantly better on the rotarod compared to mice on the control diet independent of genotype. We did not observed KD-induced changes in spatial or associative memory in the radial arm water maze or contextual fear conditioning, respectively. Furthermore, immunohistochemical levels of amyloid, tau, astrocytic and microglial markers showed no differences between animals fed KD or the control diet. Chapter 3 studied the effects of calorie restriction on a mouse model of tau deposition. We show here that 35% body weight reduction in Tg4510 mice did not prevent increased locomotor activity in the open field, previously reported in chapter 2. Similarly, CR did not affect motor performance or spatial memory assessed by the rotarod and radial arm water maze, respectively. Interestingly, CR Tg4510 mice showed improved short-term memory tested by the novel object recognition despite spending a minimal percentage of the trial time interacting with the objects presented. However, this improvement was not observed when the test was modified to replace the objects with mice. In this case, we noticed that nontransgenic mice spent most of the trial time interacting with the novel mouse whereas Tg4510 mice spent roughly the same amount of time at any of the areas in the test chamber. Moreover, no changes in histopathological or biochemical levels of tau, astrocytic, microglial or synaptic markers were observed. Chapter 4 sought to investigate alternative approaches to inducing ketosis in the brain by either administering BHB intracerebroventricularly (i.c.v.) or by using the acetoacetate (AcAc) diester as a dietary supplement in mice. We observed that i.c.v administration of BHB in 20 months old APP mice did not affect body weight or food intake. Consistent with the lack of effects on behavioral performance, amyloid and congophilic load were not different between APP mice infused with either saline or BHB. We also found that enteral administration of AcAc diester was well tolerated and induced peripheral ketosis for at least 3 hours. Acute ketosis, however, was not sufficient to attenuate behavioral deficits in old APP mice. Chronic dietary supplementation with AcAc was tested in control tet mice and was shown to effectively induce ketosis in mice fed a diet with normal contents of carbohydrates. Nonetheless, we observed that AcAc-induced ketosis was not significantly greater than levels induced by the ketogenic diet tested in our lab. Considering that KD did not rescue behavioral or histopathological features of either amyloid or tau depositing mouse models, we anticipated that dietary supplementation with AcAc would not likely modify the phenotype of the same mouse models tested previously. Taken together, our findings show that our custom made ketogenic diet was effective in inducing and sustaining ketosis and may play an important role in enhancing motor performance in mice. However, the lack of changes on the cognitive and histopathological phenotype of the models studied suggests that KD may not be a disease modifying therapeutic approach to AD. Moreover, calorie restriction showed inconsistent effects on behavioral and histopathological outcomes of a mouse model of tauopathies. Furthermore, dietary supplementation with acetoacetate diester was successful in inducing peripheral ketosis to the same extend as a ketogenic diet even in the context of normal carbohydrate intake, suggesting that it may be of therapeutic interest for diseases of hypometabolism but not a disease modifying therapy in mouse models of Alzheimer's pathology.

Succinic semialdehyde dehydrogenase (ALDH5A1) deficiency (SSADH-d) is an autosomal recessive, inborn error of gamma-aminobutyric acid (GABA) metabolism that results in psychomotor retardation, ataxia and seizures. A mouse model of SSADH-d (the Aldh5a1-/- mouse) was created to study the pathophysiology and treatment of SSADH-d. Aldh5a1-/- mice have psychomotor retardation and a progressive seizure phenotype results in death around P25. The present experiments tested the effects of a ketogenic diet in the treatment of Aldh5a1-/- mice. The KD was found to prolong the lives of Aldh5a1-/- mice by >300% while significantly delaying the onset the ataxia and preventing weight loss that is seen in untreated Aldh5a1-/- mice. Electrophysiological recordings revealed a corresponding decrease in seizures in KD fed mutants, as compared to control diet (CD) fed mutants. We assessed spontaneous miniature postsynaptic currents (mPSC) in CD and KD fed mutants. We found that CD fed mutants had significantly decreased inhibitory mPSC (mIPSC) activity compared to CD fed wildtype controls. mIPSC activity was restored in KD fed Aldh5a1-/- mice. A similar effect was found in [35S]TBPS binding experiments. TBPS binding was significantly reduced in CD fed Aldh5a1-/- mice, but restored in KD fed mutants. Plasma analysis revealed that an elevation of serum beta-hydroxybutyrate may play a role in the KD’s effects. The KD led to a significant elevation in the number of hippocampal mitochondria in mutant mice. Further, the KD was able to normalize the deficiencies in the hippocampal ATP levels seen in the Aldh5a1-/- mice. The present data suggest that the KD is able to significantly improve the Aldh5a1-/- phenotype. The effect of the KD on mIPSC activity is novel and furthers our understanding of how the KD may exert its effects. The mitochondrial studies confirm the findings of others, that the KD elevates the number of mitochondria. The KD also restores ATP deficiencies in Aldh5a1-/- mice, which is a novel finding. Together, these show that the KD may be an effective treatment for SSADH-d in humans. These data also further our understanding of the KD’s mechanisms of action.

Succinic semialdehyde dehydrogenase (ALDH5A1) deficiency (SSADH-d) is an autosomal recessive, inborn error of gamma-aminobutyric acid (GABA) metabolism that results in psychomotor retardation, ataxia and seizures. A mouse model of SSADH-d (the Aldh5a1-/- mouse) was created to study the pathophysiology and treatment of SSADH-d. Aldh5a1-/- mice have psychomotor retardation and a progressive seizure phenotype results in death around P25. The present experiments tested the effects of a ketogenic diet in the treatment of Aldh5a1-/- mice. The KD was found to prolong the lives of Aldh5a1-/- mice by >300% while significantly delaying the onset the ataxia and preventing weight loss that is seen in untreated Aldh5a1-/- mice. Electrophysiological recordings revealed a corresponding decrease in seizures in KD fed mutants, as compared to control diet (CD) fed mutants. We assessed spontaneous miniature postsynaptic currents (mPSC) in CD and KD fed mutants. We found that CD fed mutants had significantly decreased inhibitory mPSC (mIPSC) activity compared to CD fed wildtype controls. mIPSC activity was restored in KD fed Aldh5a1-/- mice. A similar effect was found in [35S]TBPS binding experiments. TBPS binding was significantly reduced in CD fed Aldh5a1-/- mice, but restored in KD fed mutants. Plasma analysis revealed that an elevation of serum beta-hydroxybutyrate may play a role in the KD’s effects. The KD led to a significant elevation in the number of hippocampal mitochondria in mutant mice. Further, the KD was able to normalize the deficiencies in the hippocampal ATP levels seen in the Aldh5a1-/- mice. The present data suggest that the KD is able to significantly improve the Aldh5a1-/- phenotype. The effect of the KD on mIPSC activity is novel and furthers our understanding of how the KD may exert its effects. The mitochondrial studies confirm the findings of others, that the KD elevates the number of mitochondria. The KD also restores ATP deficiencies in Aldh5a1-/- mice, which is a novel finding. Together, these show that the KD may be an effective treatment for SSADH-d in humans. These data also further our understanding of the KD’s mechanisms of action.

abstract: Patients with malignant brain tumors have a median survival of approximately 15 months following diagnosis, regardless of currently available treatments which include surgery followed by radiation and chemotherapy. Improvement in the survival of brain cancer patients requires the design of new therapeutic modalities that take advantage of common phenotypes. One such phenotype is the metabolic dysregulation that is a hallmark of cancer cells. It has therefore been postulated that one approach to treating brain tumors may be by metabolic alteration such as that which occurs through the use of the ketogenic diet (KD). The KD is high-fat, low-carbohydrate diet that induces ketosis and has been utilized for the non-pharmacologic treatment of refractory epilepsy. It has been shown that this metabolic therapy enhances survival and potentiates standard therapy in mouse models of malignant gliomas, yet the anti-tumor mechanisms are not fully understood. The current study reports that KetoCal® (KC; 4:1 fat:protein/carbohydrates), fed ad libitum, alters hypoxia, angiogenic, and inflammatory pathways in a mouse model of glioma. Tumors from animals maintained on KC showed reduced expression of the hypoxia marker carbonic anhydrase 9 (CA IX), a reduction in hypoxia inducible factor 1-alpha (HIF-1&#945;) and decreased activation of nuclear factor kappa B (NF-&#954;B). Animals maintained on KC also showed a reduction in expression of vascular endothelial growth factor receptor 2 (VEGFR2) and decreased microvasculature in their tumors. Further, peritumoral edema was significantly reduced in animals fed the KC and protein analysis showed significantly altered expression of the tight junction protein zona occludens-1 (ZO-1) and the water channeling protein aquaporin-4 (AQP4), both of which have been implicated in malignant processes in glioma, including the formation of peritumoral edema in patients. Taken together the data suggests that KC alters multiple processes involved in malignant progression of gliomas. A greater understanding of the effects of the ketogenic diet as an adjuvant therapy will allow for a more rational approach to its clinical use. / Dissertation/Thesis / Masters Thesis Biology 2014

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