Age-related neurodegenerative proteinophaties, including polyglutamine (polyQ) diseases such as Huntington’s disease, are a group of disorders in which a single protein or a set of proteins misfold and aggregate resulting in a progressive and selective loss of anatomically or physiologically related neuronal systems. Despite evidence showing a clear relationship between mitochondrial dysfunction, aging and neurodegenerative proteinophaties, the extent of the mitochondrial respiratory chain deficits, the involvement of mitochondrial dysfunction and the mechanisms responsible for these processes are largely unknown. Using yeast models of cellular aging and polyQ disorders we show that mitochondrial dysfunction is an important contributor to the process of aging and age-related neurodegenerative diseases. Preserving mitochondrial function is essential for standard wild-type aging. Enhancement of mitochondrial biogenesis ameliorates polyQ cytotoxicity and is a required component of interventions that retard the aging process.

The cause of amyotrophic lateral sclerosis (ALS), a cruel neurodegenerative disease, remains unclear. Trans-activating response region (TAR) DNA-binding protein of 43 kDa (TDP-43) has been suggested to have an important role in ALS pathogenesis. In this thesis, we show that a disease linked mutation in matrin 3 (MATR3), a DNA/RNA-binding protein, corresponds to an increased tendency for TDP-43 to aggregate into large and more numerous cytoplasmic inclusions that are the hallmark of ALS. Immunocytochemistry experiments show that MATR3 colocalizes with TDP-43 in vitro. These experiments also show TDP-43 is a component of both MATR3 granules and stress granules, and that MATR3 inclusions are directly adjacent to stress granules or eIF3α inclusions. We hypothesize that, while not being a part of stress granule complex, MATR3 granules are involved in RNA processing via the stress granule pathway by relaying crucial components such as TDP-43. We have also found that compound 8J is able to disaggregate and relocate TDP-43 and MATR3 positive inclusions in vitro. While the mechanism of action of compound 8J remains unclear, fluorescence activated cell sorting (FACS) experiment showed that there was a significant increase in viability in double wild type (matrin 3 and TDP-43) cells when treated with C8J (p-value <.001), which suggests that the TDP-43 and MATR3 cytoplasmic inclusions that were previously observed have a net cytotoxic effect. Together with the in vitro result on C8J, this result also suggests that C8J enhances the survivability of cells by restoring TDP-43 back to the nucleus. MATR3 biochemistry seems to connect to neurodegenerative diseases in several ways. Identifying the pathological connections between MATR3 and TDP-43 physiology will provide us with a greater understanding of ALS pathology.

The Hippo pathway is a cell signaling pathway involved in organ size regulation and tumorigenesis in mammals. This pathway regulates the activity of Yes-associated protein (YAP), a transcriptional coactivator which binds to the transcription factor TEAD to promote expression of genes controlling growth and proliferation of tissues, as well as inhibition of apoptosis. The Hippo pathway has recently been implicated as a pathogenic mechanism in neurodegenerative disorders. Specifically, mammalian sterile 20 (Ste20)-like kinase 1 (MST1), a protein kinase in the Hippo pathway, has been found to promote neuronal death under conditions of oxidative stress. Moreover, homozygous deletion of MST1 in a mouse model of Amyotrophic Lateral Sclerosis (ALS) significantly delayed onset of neurodegenerative symptoms. We examined the expression levels of key Hippo pathway components in cortex, lumbar spinal cord, and gastrocnemius muscle samples of male and female G39A SOD1 mice using western blots. Our results revealed a significant increase in phosphorylated MST1 (pMST1) in lumbar spinal cord of presymptomatic transgenic animals, and found this increase to be sex and gene copy number dependent. These results suggest that the Hippo pathway is dysregulated in the SOD1 mouse model and that MST1 may play a critical role in pathogenesis and disease progression in ALS.

L'autophagie est l'une des voies de signalisation qui maintiennent l'homéostasie cellulaire en condition basale, mais aussi en réponse à un stress. Son rôle est essentiel pour assurer plusieurs fonctions physiologiques, et son altération est associée à de nombreuses maladies, parmi lesquelles le cancer, les maladies immunitaires et les maladies neurodégénératives. Un nombre croissant d'études a établi que la voie autophagique est finement contrôlée. Cependant, très peu est connu sur les mécanismes moléculaires assurant sa régulation mais la famille des E3-ligases joue un rôle primordiale. La Gigaxonine est un adaptateur de la famille des E3 ligases CUL3, qui spécifie les substrats pour leur ubiquitination et leur successive dégradation. Des mutations «perte de fonction» de la Gigaxonine causent la Neuropathie à Axones Géants (NAG), une maladie neurodégénérative sévère et fatale, qui impacte tout le système nerveux et provoque une agrégation anormale des Filaments Intermédiaires (FI) dans l'organisme entier. Grâce à la modélisation de la pathologie dans les cellules de patients et chez la souris, le laboratoire a pu mettre en avant le rôle crucial de la Gigaxonine dans la dégradation de la famille des FIs, à travers son activité d'ubiquitination.Au cours de ma thèse, j'ai étudié les mécanismes de neurodégénerescence de la NAG, et la possible altération de la voie autophagique.Pour cela, j'ai développé un nouveau modèle neuronal de la maladie, à partir de notre modèle murin NAG, qui reproduit la mort neuronale et l'agrégation des FIs retrouvées chez les patients. Pour étudier l'implication de l'autophagie dans la neurodégénérescence, j'ai évalué l'effet de la déplétion de la Gigaxonine sur la formation des autophagosomes, le flux autophagique, la fusion avec le lysosome et la dégradation. J’ai ainsi révélé un défaut dans la dynamique autophagique dans les neurones NAG -/-. Pour déchiffrer les mécanismes moléculaires sous-jacents, j'ai étudié l'effet de l'absence de la Gigaxonine sur différentes régulateurs de la voie. En utilisant des techniques complémentaires, j'ai montré que la Gigaxonine est essentielle pour le turn-over d’un interrupteur autophagique, à travers son activité d’E3-ligase.En conclusion, nous avons identifié un nouveau mécanisme moléculaire impliqué dans le contrôle des premières phases de l'autophagie. Non seulement ces résultats présentent une avancée significative dans le domaine de l'autophagie, ils contribuent également à la compréhension de son dysfonctionnement dans les maladies neurodégénératives, et pourraient générer une nouvelle cible pour une intervention thérapeutique chez l'homme. / The autophagic route is one of the signaling pathways that sustain cellular homeostasis in basal condition, but also in response to stress. It has been shown to be crucial for several physiological functions and its impairment is associated with many diseases, including cancer, immune and neurodegenerative diseases. While an expanding number of studies have shown that autophagic route is finely controlled, little is known about the molecular mechanisms ensuring its function, but a fundamental role is sustained by the family of E3 ligases. Gigaxonin is an adaptor of a Cul3-E3 ligase, which specifies the substrates for their ubiquitination and their subsequent degradation. “Loss of function” mutations in Gigaxonin cause Giant Axonal Neuropathy (GAN), a severe and fatal neurodegenerative disorder that impacts broadly the nervous system and cause an abnormal aggregation of Intermediate Filaments (IFs) through the body. Modeling the disease in patient’s cells and in mouse, the laboratory has demonstrated the crucial role of Gigaxonin in degrading the entire family of IFs through its ubiquitination activity.During my PhD, I studied the neurodegenerative mechanisms in GAN disease, and the possible impairment of autophagy pathway.For that purpose, I developed a new neuronal model of the disease from our GAN mouse, which reproduced the neurodegeneration and the IF aggregation found in patients. To investigate the involvement of autophagy in neurodegeneration, I evaluated the effect of Gigaxonin depletion on autophagosome formation, autophagic flux, lysosome fusion and degradation, and I revealed a defect in autophagy dynamics. To decipher the molecular mechanism of autophagosome impairment, I investigated the effect of Gigaxonin depletion on different autophagy regulators. Using complementary techniques, I showed that Gigaxonin is essential for the turn-over of a specific molecular switch, through its E3 ligase activity.Altogether, we identified a new exciting molecular mechanism in the control of autophagy. Not only these findings present a significant advance in the comprehension of the fundamental field of autophagy, but it also contribute in the understanding of its dysfunction in neurodegenerative diseases, and may generate a new target for therapeutic intervention in humans.

Learning and memory in adult females decline during menopause and estrogen replacement therapy is commonly prescribed during menopause. Post-menopausal women tend to suffer from depression and are prescribed antidepressants – in addition to hormone therapy. Estrogen replacement therapy is a topic that engenders debate since several studies contradict its efficacy as a palliative therapy for cognitive decline and neurodegenerative diseases. Signaling transduction pathways can alter brain cell activity, survival, and morphology by facilitating transcription factor DNA binding and protein production. The steroidal hormone estrogen and the anti-depressant drug lithium interact through these signaling transduction pathways facilitating transcription factor activation. The paucity of data on how combined hormones and antidepressants interact in regulating gene expression led me to hypothesize that in primary mixed brain cell cultures, combined 17beta-estradiol (E2) and lithium chloride (LiCl) (E2/LiCl) will alter genetic expression of markers involved in synaptic plasticity and neuroprotection. Results from these studies indicated that a 48 h treatment of E2/LiCl reduced glutamate receptor subunit genetic expression, but increased neurotrophic factor and estrogen receptor genetic expression. Combined treatment also failed to protect brain cell cultures from glutamate excitotoxicity. If lithium facilitates protein signaling pathways mediated by estrogen, can lithium alone serve as a palliative treatment for post-menopause? This question led me to hypothesize that in estrogen-deficient mice, lithium alone will increase episodic memory (tested via object recognition), and enhance expression in the brain of factors involved in anti-apoptosis, learning and memory. I used bilaterally ovariectomized (bOVX) C57BL/6J mice treated with LiCl for one month. Results indicated that LiCl-treated bOVX mice increased performance in object recognition compared with non-treated bOVX. Increased performance in LiCl-treated bOVX mice coincided with augmented genetic and protein expression in the brain. Understanding the molecular pathways of estrogen will assist in identifying a palliative therapy for menopause-related dementia, and lithium may serve this purpose by acting as a selective estrogen-mediated signaling modulator.

Indiana University-Purdue University Indianapolis (IUPUI) / In glaucoma, the connection between the eye and the brain is severed leading to the degeneration of retinal ganglion cells (RGCs) and eventual blindness. A need exists to better understand the maturation of human RGCs as well as their degeneration, with the goal of developing new therapeutics diseases like glaucoma. Human pluripotent stem cells (hPSCs) provide an advantageous model for the study of RGC development and disease as they can be differentiated into RGCs in large, reproducible quantities. Efforts of the current studies initially focused on the development and maturation of RGCs from hPSCs. RGCs derived from hPSCs were a diverse population of cells and matured in a temporal fashion, yielding morphological and functional characteristics similar to their in vivo counterpart. CRISPR/Cas9 gene editing was then utilized to insert the OPTN(E50K) glaucomatous mutation into hPSCs to model RGC degeneration. RGCs harboring this mutation exhibited numerous degenerative phenotypes including neurite retraction an autophagy dysfunction. Within the retina, many cell types contribute to the health and maturation of RGCs including astrocytes. As such, a co-culture system of hPSC-derived RGCs and astrocytes was developed to better understand the interaction between these two cell types. When grown in co-culture with astrocytes, hPSC-derived RGCs demonstrated significantly enhanced and accelerated morphological and functional maturation, indicating an important relationship between these cells in a healthy state. Astrocytes have also been shown to encompass neurodegenerative phenotypes in other diseases of the CNS, with these deficits profoundly effecting the health of surrounding neurons. hPSC-derived astrocytes grown from OPTN(E50K)-hPSCs demonstrated cell autonomous deficits and exhibited significant effects on the degeneration of RGCs. Taken together, results of this study demonstrated the utilization of hPSCs to model RGC maturation and degeneration in glaucoma. More so, these results are one of the first to characterize astrocyte deficits caused by the OPTN(E50K) mutation and could provide a new therapeutic target for pharmacological screenings and cell replacement therapies to reverse blindness in optic neuropathies.

Indiana University-Purdue University Indianapolis (IUPUI) / Cerebral amyloid angiopathy (CAA) is characterized by the cerebrovascular deposition of amyloid. The mechanisms underlying the contribution of CAA to neurodegeneration are not fully understood. In this dissertation, there are three main chapters. The first chapter investigates existing evidence regarding the amyloid diversity in CAA and its relation to tau pathology and immune response, as well as the possible contribution of molecular and cellular mechanisms, previously associated with parenchymal amyloid in Alzheimer disease (AD) and AD-related dementias, to the pathogenesis of CAA. The second chapter demonstrates differential glial reactivity and activation associated with early-stage CAA in a mouse model of Familial Danish Dementia (FDD), a neurodegenerative disease characterized by vascular accumulation of Danish amyloid (ADan). We show that early-stage CAA is associated with dysregulation in immune response networks and lipid processing, severe astrogliosis with a neurotoxic A1-astrocytic phenotype, characterized by increased expression of Complement Component 3 (C3), and decreased levels of Triggering Receptor Expressed On Myeloid Cells 2 (Trem2) with no significant reactive microgliosis. Our results also indicate how cholesterol accumulation and Apolipoprotein E (ApoE) are associated with vascular amyloid deposits at the early stages of pathology. Furthermore, we demonstrate A1 astrocytic mediation of Trem2 and microglia homeostasis. In the final chapter, we addressed whether inflammatory stimulus of other cell types are capable of inducing a subtype of neurotoxic astrocytes. Here we show a subtype of C3+ neurotoxic astrocyte are induced by activated endothelial cells that is distinct from astrocytes classically activated by microglia. We show that endothelial activated astrocytes have upregulated expression of A1-astrocytic genes and exhibit a distinctive extracellular matrix remodeling profile. Finally, we demonstrate that endothelial activated astrocytes are Decorin-positive and are associated to vascular amyloid deposits but not parenchymal amyloid plaques in mouse models and AD/CAA patients. These findings show the existence of potentially extensive and subtle functional diversity of C3+-reactive astrocytes.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer’s disease (AD) is a neurodegenerative disorder resulting in progressive memory loss, brain atrophy, and eventual death. AD pathology is characterized by the accumulation of neurotoxic amyloid-beta (Aβ) plaques, synapse loss, neurofibrillary tangles (NFTs), and neurodegeneration. The APOE4 allele is associated with a 3-fold increased risk for AD and results in increased Aβ plaque deposition, reduced Aβ clearance, and reduced synaptic plasticity. Although APOE expression is upregulated in microglia in AD, APOE is expressed primarily by astrocytes in the CNS. It is not well understood how astrocytic APOE drives the mechanisms that result in worsened AD outcomes. Here, digital spatial profiling and bioinformatics data suggest that APOE4 causes transcriptional dysregulation in early AD and may disrupt neuronal processes via astrocytes. Whole transcriptome data from plaque and non-plaque regions in the cortices and hippocampus of 4- and 8-month-old AD model mice expressing humanized APOE4/4 or APOE3/3 (control) were analyzed. Transcriptional dysregulation was increased in APOE4/4 AD mice compared to that in APOE3/3 at 4 but not 8 months of age, suggesting that early dysregulation of APOE4-driven disease mechanisms may shape degenerative outcomes in late-stage AD. Additionally, APOE4/4 potentially functions via plaque-independent mechanisms to influence neuronal function in early AD before the onset of pathology. Single-nuclei RNA sequencing data were obtained from human post-mortem astrocytes and the bioinformatic analyses revealed a novel astrocyte subtype that highly expresses several top genes involved in functional alterations associated with APOE4, including neuronal generation, development, and differentiation, and synaptic transmission and organization. Overall, our findings indicate that APOE4 may drive degenerative outcomes through the presented astrocyte candidate pathways. These pathways represent potential targets for investigations into early intervention strategies for APOE4/4 patients. / 2024-05-22

Huntington's disease (HD) is an inherited autosomal-dominant neurodegenerative disease caused by the abnormal expansion of CAG repeats in exon 1 of the huntingtin gene located on chromosome 4. This disease is characterized by the premature loss of medium spiny neurons in the striatum and behavioural deficits that typically manifest at middle-age. Despite the identification of its cause decades ago, there is still no disease modifying treatment available for HD patients. Current evidence indicates that exacerbated glutamate signalling in the striatum plays a key role in the pathophysiology of HD. Within the striatum, metabotropic glutamate receptor (mGluR) 2/3 are predominantly expressed on presynaptic terminals, whereas mGluR5 is predominantly localized to postsynaptic terminals. Here, we show that both the activation of mGluR2/3 and the inhibition of mGluR5 can improve HD symptoms in the zQ175 HD mouse model. Specifically, treating zQ175 HD mice with either the mGluR2/3 agonist LY379268 or the mGluR5 negative allosteric modulator (NAM) CTEP rescues motor deficits, reduces mutant huntingtin aggregate formation, improves neuronal survival and alleviates microglia activation. We also provide evidence that shows sex can influence the progression of HD symptoms and the efficacy of therapeutic agents. We found that chronic administration of LY379268 differentially activated and inactivated cell signalling pathways in male and female zQ175 mice. Furthermore, female zQ175 mice required a longer treatment duration with CTEP than male mice to show improvement in their rotarod performance. Using FDNQ175 mice, a newer HD mouse model derived from the zQ175 line, we demonstrated that female FDNQ175 mice were less susceptible to decline in limb function than male mice but showed higher levels of insoluble mutant huntingtin aggregates at a younger age.