Autophagy: Cellular Homeostasis

Huntingtin is a 350-kilodalton protein of unknown function that is mutated in Huntington's disease (HD), a neurodegenerative disorder. The mutant protein is presumed to acquire a toxic gain of function that is detrimental to striatal neurons in the brain. However, loss of a beneficial activity of wild-type huntingtin may also cause the death of striatal neurons. Here we demonstrate that wild-type huntingtin up-regulates transcription of brain-derived neurotrophic factor (BDNF), a pro-survival factor produced by cortical neurons that is necessary for survival of striatal neurons in the brain. We show that this beneficial activity of huntingtin is lost when the protein becomes mutated, resulting in decreased production of cortical BDNF. This

The HEXA gene provides instruction for making part of an enzyme called beta-hexosaminidase A, which plays a very critical role in the brain and spinal cord. This enzyme is found in the lysosomes, which are the structures in cells that break down toxic substances and their job is that of a recycling center. Beta-hexosaminidase A helps break down a fatty substance called GM2 ganglioside. The mutations in the HEXA gene disrupt the activity of beta-hexosaminidase A, which prevents the enzyme from breaking down GM2 ganglioside. As a result, this substance accumulates to toxic levels, particularly in the neutrons in the brain and also the spinal cord. Damage by the buildup of GM2 ganglioside leads to the destruction of the neurons; Causing signs and symptoms of Tay-Sachs disease.

The changes that occur in the niche are more important than in stem cells themselves when it comes to the failure of stem cell activity with aging. (Fowler, 2012)

Neurodegenerative diseases continue to affect the lives of millions Americans each year, with incidence and prevalence rates ever increasing. These diseases cause degeneration or death of nerve cells in the brain. These diseases can cause a financial and emotional burden on not only patients themselves, but also family members and care givers as well. Molecular mechanisms that underlie these diseases have remained relatively unclear, despite much research. Understanding the mechanisms of these diseases are facilitated by utilizing model organisms to study pathways involved in neurodegenerative diseases. One such model organism is the Caenorhabditis elegans nematode. The C. elegans roundworm has displayed usefulness as a template to study neurodegenerative diseases in humans, including Parkinson’s disease and Alzheimer’s disease.

GnT-III suppression alters Notch receptor localization- Autophagy has been shown to regulate the Notch pathway in normal physiology and disease (16). Therefore we performed immunocytochemistry to determine the localization of Notch 1 in OVCAR3 cells. Notch 1 is observed in intracellular vesicles and the nucleus in control shRNA OVCAR3 cells and does not colocalize with LAMP1 (Fig. 5A). However, in GnT-III shRNA OVCAR3 cells Notch 1 is primarily localized with the late endosome/lysosome marker LAMP1 indicating that a larger percentage of Notch 1 is in this fraction (Fig. 5A, white arrows). To further confirm these findings we treated cells with chloroquine to raise the pH of this cellular compartment and inhibit lysosomal enzymes (17). We found no effect on Notch 1 levels in control cells treated with chloroquine; while GnT-III shRNA cells show a significant increase in Notch 1 levels when lysosomal enzymes are inhibited (Fig. 5B).

The concept of neurogenesis being confined to the embryonic stage became less obvious with the onset of discovery of neural stem cells maintained in two distinct regions of the mammalian adult brain namely dentate gyrus (DG) of the hippocampus and the sub ventricular zone (SVZ) of the forebrain lateral ventricles14,18,19. What makes these neural stem cells a more credible target for oncogenic transformation? The continual presence of undifferentiated, mitotically active, self renewable stem cells at the apex of the hierarchy bundled in discrete germinal niches in the mammalian brain throughout the lifespan of an organism allows them to accumulate mutations, thus rendering them vulnerable for neoplastic reprogramming. There has been increasing evidence that the genetic and epigenetic mechanisms that initiate and maintain the NSC developmental state are possibly deregulated in GB to emerge as Glioma initiating cells or Brain tumor stem cells20. The discovery of BTSC has high clinical significance in the neuro-oncology field, as evidenced by major diverse roles it plays in various aspects of tumor growth such as tumor initiation, maintenance, progression, angiogenesis and tumor recurrence owing to therapeutic resistance, some of which are described

Neurological disorders such as Alzheimer’s disease, autism spectrum disorders (ASD), and schizophrenia impact behavior and cognitive processes. While the symptoms for these diseases vary, they share disruptions in synapse development and spine formation. In the mammalian nervous system, dendritic spines are small, dynamic protrusions on neurons that develop in response to increased presynaptic activity. Although spine dynamics throughout development have been well studied, very little is known about the genes involved in spine formation. A recent study in our laboratory discovered spiny projections on a group of inhibitory neurons called DD neurons in C. elegans. Using C. elegans as a model system, the goal of this project is to identify genes responsible for dendritic spine formation. Specifically, I will 1) conduct a forward genetic screen and examine candidate axon guidance molecules to identify genes involved in dendritic spine formation, and 2) examine the effect hyper- and hypo-presynaptic activity has on the development of spines using locomotory changes, such as thrashing and microfluidic assays, and cell-specific changes using channel rhodopsin and temperature sensitive alleles. Given the strong homology between C. elegans and mammalian genes, this research will help identify conserved mechanisms underlying spine formation and maintenance. Ultimately, understanding spine development will help shape the treatment and prevention of

Since mature autophagosome fuse with lysosomes to become autolysosome in the very late stage of autophagy, we asked whether miR34a could retard autophagosome-lysosome fusion by examining the co-localization of EGFP-LC3 and LysoTracker Red, a specific dye for live cell lysosome labeling [47]. As expected, EGFP-LC3 puncta were dramatically reinforced in miR34a mimic transfected cells but did not co-localize with LysoTracker Red. This was acting similar to cells treated with bafilomycin A1 (Baf), which blocked autophagosome-lysosome fusion. In contrast, E64D treatment induced a remarkable increased EGFP-LC3 puncta, which were well co-localized with LysoTracker Red (Fig. 1D). Since lysosome-associated membrane glycoprotein 2 (LAMP2) is also critical for autophagosome-lysosome fusion [48], we examined the effect of miR34a on LAMP2 expression using WB analysis. MiR34a caused an unexpected increase of LAMP2 (Supplementary Fig. 1C), suggesting that the mechanism by which miR34a blocked autophagosome-lysosome fusion was not due to LAMP2. As LAMP2 is mostly important for chaperone mediated autophagy, its increase might be a compensation for the decrease in macroautophagy. In summary, the above results reinforce that miR34a compromised Aβ degradation by, at least, blocking autolysosome formation.

Age is known to be one of the contributing factors that seem to play a role in the development of Alzheimer’s disease. Human neurons are functioning cells of the nervous system that operate over an entire lifetime, yet the mechanisms that help preserve and protect against neurodegeneration during aging are unknown. There are fundamental questions as to why some individuals age with their cognitive functions relatively intact and others who decline and develop Alzheimer’s disease. Scientist have made it clear that neuronal cell numbers is preserved our neurocortex and hippocampus of the aging human brain and declining only in the setting stages of this type of disease. Robust stress-response mechanism (REST) is a repressor of embryonic development

These proteins are expressed by oligodendrocytes and Schwann cells and act to inhibit the outgrowth of neurites. A recent review by Geoffroy and Zheng discusses the biology of these signaling pathways and their significance in CNS injury [18]. Membrane-associated glycoprotein (Mag) is a component of the immunoglobulin superfamily of proteins [19], and is located mainly on the periaxonal loop of the myelin sheath [20], where it is capable of contacting neurons. Mag primarily acts as a potent inhibitor of sprouting [21, 22], but can exhibit a stimulatory function on embryonic neurons [23]. Nogo-A, a member of the reticulon family of proteins [24], is expressed preferentially in oligodendrocytes and represents another strong inhibitor of neurite outgrowth [25]. The localization of Nogo-A within oligodendrocytes is noteworthy. Like other reticulons, it is enriched on the endoplasmic reticulum, a location that would normally be inaccessible to receptors on sprouting neurons. However, it is possible that this reservoir of Nogo-A could become active specifically upon cellular damage to the oligodendrocyte, which would expose it to the extracellular space [17]. Nogo-A is also expressed on the inner myelin membrane, similar to Mag [25]. Finally, oligodendrocyte myelin glycoprotein (Omgp) is a minor component of the myelin sheath that localized near the

iPSC’s were differentiated into neural stem cells by using SMAD-pathway inhibition. Green fluorescent proteins were labeled iPSC, and then a “serum- free floating culture of embryoid body-like aggregates method” was combined with the SMAD- pathway. These methods stimulated the LIF/BMP signaling. Leukemia inhibitory factor and bone morphogenic protein both promote neural cell differentiation into GFAP (Glial

Autophagy is a evolutionarily conserved essential proteostasis and catabolic membrane trafficking process that has close associations with health and longevity [10, 11]. It maintains cellular homeostasis and viability by regulating the quantity and quality of cytoplasmic constituents such as organelles and macromolecules through lysosomal degradation, which is activated in response to nutrient starvation and other stressors to generate energy and allow survival [12]. Autophagy plays vital roles by self-degrading under a variety of physiological and pathological conditions such as maintaining the stem cell quiescent state [13], manipulating immunity [14], regulating bone growth [15], carcinogenesis [16-18] and cell metabolism [18]. Importantly, Autophagy directly targets pathogenic misfolded Aβ for degradation [19]. Chemical that enhances autophagy was shown to be capable of restoring nesting behavior in a murine model of Alzheimer disease [20]. Extensive efforts have been made on Aβ degradation by autophagy, though people also argued that

Homeostasis must be maintained in organisms in order for enzymes to function properly. It must be maintained because if organisms have sub-optimal enzyme function, they won’t perform the correct functions. Homeostasis helps enzymes perform how they are supposed to because enzymes dissolve and break down substances, so with the change in conditions and homeostasis, your body will not absorb all the vitamins and nutrients your food gets you so you will start having less and less energy. This happens because without homeostasis, they are not able to reach the energy they need to perform. So when you have a high fever or altered blood pH, then enzyme activity would slow down in cells.

Mitochondrial respiration is the final process of metabolic reactions to generate biological energy (ATP) along with glycolysis and Krebs cycle. Mitochondrial respiration is critical not only host immune cells to limit the IVI but also influenza virus to replicate the genes for survival by exploiting energy source. Interestingly, influenza virus leads hypoactive mitochondrial respiration in epithelial cells but causes hyperactive mitochondrial respiration in immune cells, especially macrophages, so that stimulates the generation of ROS and antiviral signaling pathways. Thus, regulation of mitochondrial respiration in alveolar macrophages can be a potential target of IVI. Recent evidences suggested that miRNAs can modulate mitochondrial respiration

UPS is a major pathway that plays an integral role in abetting the clearance of damaged, misfolded and senescent proteins from nucleus and cytoplasm to maintain tissue homeo-stasis. However, in the neurodegenerative disorders (AD) aggregation of pathogenic proteins (Aβ) compromise UPS function and delay the degradation of proteasome substrates. Many of these substrates are essential for supporting and regulating highly dynamic connections between neurons that are necessary for cognitive functions, learning and memory. The improper functioning of UPS results in inadvertent stabilization of these substrates resulting in loss of synaptic connections and diminished cognitive functions in neurodegenerative disorders. Therefore to maintain cellular homeostasis it is essential to keep the ubiquitin-dependent proteolysis operative as deficits in the UPS system result in accumulation of proteasome substrates. But, so far there is no evidence whether UPS exert any control on the expression of proteins that regulate the transition from proliferation to neural differentiation of stem/ progenitor cells. Further, the relationship between neurodevelopmental pathways and UPS is poorly understood. Therefore, in an effort to limit the onset and progression of AD it is imperative to understand the interdependence of UPS and neurogenesis so that the actual potential of gene regulatory networks involved in the survival and maturation of neurons in the brain can be harnessed. Overall the proposed work will provide a deep look into the cellular and molecular mechanisms responsible for maintaining the stemness characteristics in NSCs and the generation of functional neurons and how they can be modulated to provide alternative therapeutic targets to slow the cognitive impairment and behavioral disturbances associated with