Anatomy and Neuroscience - Theses
Permanent URI for this collection
Search Results
1 - 3 of 3
-
ItemImpact of lipofuscin accumulation on retinal degeneration in the mouse model of the Neuronal Ceroid Lipofuscinosis variant CLN6( 2018)Failure of the autophagy-lysosomal pathway and accumulation of lipofuscin in the retinal pigment epithelium (RPE) has been suggested to induce photoreceptor dysfunction and death in age related macular degeneration (AMD), Stargardt’s disease and neuronal ceroid lipofuscinosis (NCL). With detailed knowledge about the underlying causes and molecular mechanisms involved still elusive, mouse models of NCLs provide a useful insight into the impact of intracellular waste accumulation (liposfuscin) on cellular function. All NCL forms share the ubiquitous accumulation of lipofuscin in non-mitotic cell types such as retinal neurons and the RPE as a pathomorphological hallmark. The fundamental aim of this study was therefore to investigate the effects of lipofuscin accumulation in the naturally occurring mouse model of Battens disease, CLN6nclf, to determine the mechanisms of photoreceptor death. The time course of retinal degeneration in CLN6nclf mice was carefully characterised and correlated with changes in the intracellular waste recycling system, the autophagylysosomal pathway, in the RPE and retinal neurons. In addition, a treatment targeting the RPE called nanosecond laser therapy, which has been shown to reduce waste accumulation in the RPE in patients with AMD, was tested. Fluorescence immunohistochemistry, gross histology and twin flash electroretinography (ERG), revealed an early loss of rod photoreceptor function prior to cellular death, despite lipofuscin accumulation in all retinal cell types including the RPE. The role of the autophagy-lysosome pathway was assessed in individual retinal layers and the RPE using a novel technique for the quantification of autophagosomes and lysosomes. The results showed that rod photoreceptor death does not occur due to RPE dysfunction. Instead, rod photoreceptors showed early changes in autophagosomal-lysosomal fusion events and subsequent failure of lysosomal degradation, which resulted in specific photoreceptor dysfunction and death. Nanosecond laser treatment did not alter retinal function or retinal layer thickness in the CLN6nclf mice, suggesting that treatments targeting the photoreceptors rather than the RPE may be indicated for treatment of vision loss in NCLs. In addition to lipofuscin accumulation, changes in bio-metal levels and their intracellular distribution have also been suggested to contribute to the progression of neuronal degeneration in the CLN6 variant and retinal diseases such as AMD. However, whether changes in bio-metal distribution and concentration occur in the retina and RPE of CLN6nclf mice was unknown. To investigate this, retinal sections of CLN6nclf and control mice were assessed for bio-metal concentration using X-ray fluorescence microscopy (XFM) at the Australian Synchrotron. There were developmental changes in metal distribution and concentration in the control mouse retina with age, but no changes in metal concentration in any retinal layer or the RPE of the CLN6nclf mouse when compared to the controls. CLN6nclf mice were also treated with ZnII(atsm) and CuII(atsm) metallocomplexes that have been shown to be beneficial in different CLN6 tissues. Following oral treatment of control and CLN6nclf mice with the metallo-complexes, gross histology was used to assess individual retinal layer thickness. Treatment with zinc or copper metallo-complexes or the vehicle they were prepared in resulted in a reduction of all retinal layers compared to untreated controls, suggesting alternative treatments with improved vehicle formulations are required. Taken together these findings provide detailed evidence for the specific failure of the autophagy-lysosomal pathway, leading to an early, primary loss of rod photoreceptors in CLN6nclf mice. The study also indicates that changes in bio-metals are unlikely to contribute to retinal degeneration in CLN6nclf mice and that future treatments need to be specifically tailored to enhancing removal of intracellular waste in the photoreceptors.
-
ItemNew insights into molecular and cellular pathways of neurodegeneration in amyotrophic lateral sclerosis models( 2016)Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and paralysing neurological disorder usually fatal within 2-5 years from diagnosis. First described by Jean-Martin Charcot in the late 1860s, ALS still remains a terminal disease with no effective treatments or cure. Riluzole is the only clinically-approved drug for ALS that may extend survival by 2-3 months. Therefore, there is an urgent need to understand the underlying pathogenesis of ALS to better guide development of disease-modifying treatment strategies. This thesis investigates the molecular basis of three inter-related pathogenic mechanisms implicated in motor neuron vulnerability and loss in ALS: defective energy homeostasis; disruption of protein homeostasis and abnormal RNA homeostasis. Two leading mouse models of ALS were implemented in these studies; transgenic SOD1G93A and TDP-43A315T mice in which novel pharmacological and genetic interventions were evaluated for efficacy. To examine whether defective energy metabolism is causal or consequential in the pathological cascade of ALS, the role of the key metabolic and stress sensor; AMP-activated protein kinase (AMPK) was investigated for the first time in two ALS mouse models. AMPK activation in the spinal cord associated with symptom progression, but not onset, in SOD1G93A mice, implicating AMPK activity in mediating disease course. Conversely, AMPK inactivation occurred in spinal cord and brain of pre-symptomatic TDP-43A315T mice by a protein phosphatase 2A-dependent mechanism, identifying a novel regulation of AMPK activity by pathogenic TDP-43. AMPK inactivity may therefore drive disease initiation in this mouse model. Hence, mutant SOD1 and TDP-43 exert contrasting effects on regulation of AMPK activation which may reflect intrinsic differences in energy metabolism and neurodegeneration in these two ALS mouse models. Next, a novel pharmacological strategy to improve protein homeostasis and motor neuron health was developed and evaluated for ALS. The intracellular catabolic pathway, autophagy, particularly macroautophagy, was robustly induced in mutant SOD1 and TDP-43 models of ALS. To potentiate autophagy in ALS, a novel autophagy enhancer rilmenidine was used to stimulate mTOR-independent macroautophagy in mutant SOD1 cell and mouse models. Rilmenidine treatment achieved efficient macroautophagy induction in vitro and in vivo. However, the treatment worsened motor neuron degeneration and survival of male SOD1G93A mice by exacerbating accumulation of insoluble and misfolded SOD1 species and aggregates in spinal cords. Thus, macroautophagy stimulation using rilmenidine may mediate disease progression in this specific mouse model of ALS. Lastly, a new gene therapy strategy to alleviate defects in the RNA binding protein TDP-43 was investigated. Survival motor neuron (SMN) protein deficiency causes progressive motor neuron degeneration in spinal muscular atrophy (SMA) and may be linked to pathology in ALS. SMN overexpression was previously determined to be beneficial in mutant SOD1 models of ALS. To extend these studies to TDP-43 proteinopathy, upregulation and accumulation of endogenous SMN protein into stress granules within motor neurons was demonstrated for the first time in TDP-43A315T mice. The impact of forced SMN overexpression in TDP-43A315T mice was examined, revealing improved SMN nuclear targeting, motor neuron survival, neuroinflammation and metabolic deficits as shown by AMPK activation, in female mice. Furthermore, levels of androgen receptor (AR), mutations of which cause spinal bulbar muscular atrophy (SBMA), were significantly impaired in spinal cords of male TDP-43A315T mice. This provides evidence for shared biochemical pathways in ALS, SMA and SBMA, mediated by deficiency of factors such as SMN and AR which confer motor neuron vulnerability. In summary, in mutant SOD1-linked disease, persistent AMPK signalling and autophagy activation in motor neurons may be key determinants of disease progression. In mutant TDP-43-mediated ALS, AMPK inactivation and cytoplasmic accumulation of SMN in motor neurons may be early events triggering disease onset. In conclusion, this thesis provides novel insights into pathogenic mechanisms underlying disruption of energy, protein and RNA homeostasis within motor neurons and significant clues to therapeutic alleviation of these defective pathways in ALS. In addition, this thesis identifies new links between three main neurological disorders affecting the motor system of humans; ALS, SMA and SBMA, mediated by dysregulation of SMN and AR, suggesting shared pathogenic pathways. Finally, this work importantly extends the spectrum of motor neuron diseases that may benefit from SMN restoration, excitingly paving the way for future therapeutic development and testing of SMN enhancing agents for ALS.
-
ItemMolecular and cellular insights into mitochondrial contributions to neuronal autophagy: links to energetics and mitophagy( 2016)Neurones are essential for brain homeostasis and as highly metabolic cells rely on mitochondrial oxidative phosphorylation (OXPHOS) for energy. The integrity and functionality of mitochondria are critical for neuronal survival, and the involvement of dysfunctional mitochondria is recognized as a common theme amongst various neuropathologies. New evidence has suggested that the inappropriate clearance of dysfunctional mitochondria via autophagy (termed mitophagy) determines the pathogenesis of neurodegenerative diseases such as Parkinson’s disease. Mechanistic studies of mitophagy have been undertaken using mammalian cell lines, but this research lacks relevance to neuropathologies. This thesis investigates triggers of autophagy/mitophagy in primary neurones, and specifically if disruption of mitochondrial bioenergetics triggers neuronal autophagy, and mitophagy in particular. Cultures of primary cerebellar granule cells (CGCs) were utilized and inhibitors of the OXPHOS complexes (rotenone, 3-Nitropropionic acid, antimycin A, potassium cyanide and oligomycin targeting complex I-V, respectively), were employed to induce bioenergetic dysfunction of mitochondria. Initial investigations using MTT cell viability assay, phase contrast microscopy and cellular membrane permeabilization detected by propidium iodide staining, determined appropriate concentrations of OXPHOS inhibitors which induced effective mitochondrial damage producing slow neuronal degeneration. From this baseline adverse effects of OXPHOS inhibitors on mitochondrial bioenergetics were documented by monitoring reductions in cellular ATP level, mitochondrial membrane potential (ΔΨm) and oxygen consumption rate (OCR) of CGCs. ΔΨm was rapidly dissipated in CGCs exposed to the inhibitors of complexes I, III and IV (rotenone, antimycin A and potassium cyanide, respectively), whilst the inhibitor of complex II, 3- Nitropropionic acid, produced a much slower reduction of ΔΨm. Employing Seahorse XF24 technology allowed an incisive readout of mitochondrial functional changes where significant bioenergetic impairment was observed subsequent to inhibition of complexes I and II, which are core components of energy metabolism regulating the redox balance (NAD+/NADH levels) and TCA cycle. Existent evidence indicates depolarization of ΔΨm triggers mitophagy in mammalian cell lines, however CGCs display contrasting results where ΔΨm depolarization via complex III and IV inhibition was insufficient to elicit mitophagy despite the inducer of mitophagy, PINK1, being mobilized to mitochondria. In contrast, inhibition of complexes I and II induced mitophagy, as indicated by PINK1 mobilization and disappearance of the pH-sensitive fluorescence mitophagy reporter, mt-Rosella. Western immunoblotting of the general autophagy marker, LC3, and monitoring of acidic vesicles with monodansylcadaverine revealed activation of autophagic flux in CGCs exposed to inhibitors of complexes I-IV, indicating general autophagy in response to bioenergetic impairment irrespective of mitophagy induction. Results presented herein reveal the complexity of neuronal mitophagy and that ΔΨm may not be a necessary trigger of neuronal mitophagy. Thus inhibition of individual respiratory complexes, and notably complexes I and II, may underlie the triggering of mitophagy in primary neurones, where different mechanisms induce mitophagy in neurones compared to immortalized cell lines. This difference may be due to the unique bioenergetic dependence of neurones. Understanding the mechanisms of mitophagy and autophagy in primary neurones provides valuable insights into therapeutic approaches for neurodegenerative diseases.