Role of endolysosomes and inter-organellar signaling in brain disease

Endosomes and lysosomes (endolysosomes) are membrane bounded organelles that play a key role in cell survival and cell death. These acidic intracellular organelles are the principal sites for intracellular hydrolytic activity required for the maintenance of cellular homeostasis. Endolysosomes are involved in the degradation of plasma membrane components, extracellular macromolecules as well as intracellular macromolecules and cellular fragments. Understanding the physiological significance and pathological relevance of endolysosomes is now complicated by relatively recent findings of physical and functional interactions between endolysosomes with other intracellular organelles including endoplasmic reticulum, mitochondria, plasma membranes, and peroxisomes. Indeed, evidence clearly indicates that endolysosome dysfunction and inter-organellar signaling occurs in different neurodegenerative diseases including Alzheimer’s disease (AD), HIV-1 associated neurocognitive disease (HAND), Parkinson’s disease (PD) as well as various forms of brain cancer such as glioblastoma multiforme (GBM). These findings open new areas of cell biology research focusing on understanding the physiological actions and pathophysiological consequences of inter-organellar communication. Here, we will review findings of others and us that endolysosome de-acidification and dysfunction coupled with impaired inter-organellar signaling is involved in the pathogenesis of AD, HAND, PD, and GBM. A more comprehensive appreciation of cell biology and inter-organellar signaling could lead to the development of new drugs to prevent or cure these diseases

Role Of Aurora Kinase A, Rab4a, And Endolysosomes In The Development Of Alzheimer’s Disease-Like Pathology

Endosomes and lysosomes (endolysosomes) are organelles that play key roles in cell survival and cell death. These acidic intracellular organelles are the principal sites for intracellular hydrolytic activity required for the maintenance of cellular homeostasis. Endolysosomes are involved in the degradation of plasma membrane components, extracellular macromolecules, as well as intracellular macromolecules and cellular fragments. Understanding the physiological significance and pathological relevance of endolysosomes is now complicated by relatively recent findings of physical and functional interactions between endolysosomes and other intracellular organelles including endoplasmic reticulum, mitochondria, plasma membranes, and peroxisomes. Indeed, evidence clearly indicates that endolysosome dysfunction and inter-organellar signaling occurs in different neurodegenerative diseases including HIV-1 associated neurocognitive disease (HAND), Parkinson’s disease (PD), and Alzheimer’s disease (AD), as well as various forms of brain cancer such as glioblastoma multiforme (GBM). These findings open new areas of cell biology research focusing on understanding the physiological actions and pathophysiological consequences of inter-organellar communication. Although others and we have studied the role of endolysosomes and inter-organellar signaling in different brain diseases including HAND, PD, AD, and GBM, here, we focused on AD disease which is the most common cause of dementia. Indeed, endolysosomes are the organelles suggested to participate in early and upstream pathological signaling events that occur in the development of AD pathology. In the two studies presented here we focused on the role played by aurora kinase A (AURKA) and Rab4a proteins in the development of AD-like pathology. Alzheimer’s disease is the leading cause of dementia worldwide in people older than 65 years of age. There is currently no cure for AD and mechanisms underlying the pathogenesis of the disease are still under investigation. The pathogenesis of AD has been linked with increased levels of Aβ, p-tau, and reactive oxygen species using cellular and animal models; subcellular organelles including endolysosomes, mitochondria, and endoplasmic reticulum play key roles in AD pathogenesis. Endolysosomes are single membrane acidic compartments that contain over 60 different types of acid hydrolase enzymes. The low luminal pH of endolysosomes is an important hallmark of these organelles, which is critical for the optimum activity of pH-sensitive hydrolytic enzymes in the lumen of endolysosomes. Endolysosome dysfunction has been associated with accelerated tauopathy, amyloidogenesis, and neuritic dystrophy in the development of AD, whereas endolysosome acidification decreases amyloidogenesis and levels of p-tau. Therefore, acidifying endolysosomes may be a potential therapeutic strategy against AD pathogenesis. Based on findings that AURKA affects the activity of v-ATPase, a proton pump that plays a key role in maintaining the acidic pH of endolysosomes, and findings on decreased AURKA activity in postmortem human brain tissues, here our studies were aimed to determine the extent to which and mechanisms by which AURKA regulates endolysosome pH and amyloidogenesis. We demonstrated the presence of AURKA in primary cultured rat cortical neurons, in brains of C57BL/6J mice, and in postmortem human AD brain. AURKA phosphorylation, which correlates directly with the activity of AURKA, was decreased in human AD brain and in hippocampus of 3xTg-AD mouse brain. Using dual-excitation ratiometric measurement of endolysosome pH in neurons, we found that activation of AURKA with anacardic acid acidified endolysosomes. In SH-SY5Y cells and using pH-sensitive LysoBriteTM Red we also demonstrated that AURKA activation increased endolysosome acidification, whereas AURKA inhibition with MLN8237 de-acidified endolysosomes. AURKA activation decreased levels of Aβ, decreased the activity of BACE-1, the rate-limiting enzyme for Aβ generation, and increased the activity of cathepsin D, a lysosomal protease involved in Aβ degradation. Conversely, AURKA inhibition increased levels of Aβ and decreased the activity of cathepsin D. These findings suggest that AURKA is involved in the development of AD pathology through endolysosome-mediated actions. Our findings provide new insight into the pathogenesis of AD and highlight endolysosomes as a potential upstream therapeutic target for Alzheimer’s disease. Besides endolysosome dysfunction, endolysosome trafficking is also impaired in AD. The Rab family of small GTPases plays a critical role in regulating intracellular membrane trafficking. Early endosome enlargement is associated with upregulation of Rab4 in AD brain. However, how Rab4a dysregulation contributes to the development of AD pathology has yet to be determined. Here, we tested the hypothesis that Rab4a through endolysosome-mediated actions contributed to the development of AD-like pathology. To study the role played by Rab4a in amyloidogenesis, we generated a stable cell of SH-SY5Y cells overexpressing wild-type AβPP in which Rab4a was knocked down using shRNA lentiviral particles. We demonstrated that knocking down Rab4a expression resulted in decreased protein levels of AβPP. Moreover, we found a decrease in intracellular and secreted levels of Aβ1-40 and Aβ1-42 in Rab4a KD SH-SY5Y cells. We also found that endolysosome acidification was increased in Rab4a KD SH-SY5Y cells compared to controls. Moreover, we demonstrated that knocking down Rab4a expression resulted in decreased protein levels and activity of BACE-1, the rate-limiting Aβ generating enzyme, and increased the activity of the Aβ degrading enzyme, cathepsin D. These findings suggest that Rab4a plays a key role in the development of AD-like pathology. These studies provide new insights into understanding the mechanisms involved in the development of AD pathology and may provide us with new therapeutic strategies against AD

Involvement of Endolysosomes and Aurora Kinase A in the Regulation of Amyloid β Protein Levels in Neurons

Aurora kinase A (AURKA) is a serine/threonine-protein kinase that regulates microtubule organization during neuron migration and neurite formation. Decreased activity of AURKA was found in Alzheimer’s disease (AD) brain samples, but little is known about the role of AURKA in AD pathogenesis. Here, we demonstrate that AURKA is expressed in primary cultured rat neurons, neurons from adult mouse brains, and neurons in postmortem human AD brains. AURKA phosphorylation, which positively correlates with its activity, is reduced in human AD brains. In SH-SY5Y cells, pharmacological activation of AURKA increased AURKA phosphorylation, acidified endolysosomes, decreased the activity of amyloid beta protein (Aβ) generating enzyme β-site amyloid precursor protein cleaving enzyme (BACE-1), increased the activity of the Aβ degrading enzyme cathepsin D, and decreased the intracellular and secreted levels of Aβ. Conversely, pharmacological inhibition of AURKA decreased AURKA phosphorylation, de-acidified endolysosomes, decreased the activity of cathepsin D, and increased intracellular and secreted levels of Aβ. Thus, reduced AURKA activity in AD may contribute to the development of intraneuronal accumulations of Aβ and extracellular amyloid plaque formation

Two-pore channels regulate Tat endolysosome escape and Tat-mediated HIV-1 LTR transactivation.

HIV-1 Tat is essential for HIV-1 replication and appears to play an important role in the pathogenesis of HIV-associated neurological complications. Secreted from infected or transfected cells, Tat has the extraordinary ability to cross the plasma membrane. In the brain, Tat can be taken up by CNS cells via receptor-mediated endocytosis. Following endocytosis and its internalization into endolysosomes, Tat must be released in order for it to activate the HIV-1 LTR promoter and facilitate HIV-1 viral replication in the nucleus. However, the underlying mechanisms whereby Tat escapes endolysosomes remain unclear. Because Tat disrupts intracellular calcium homeostasis, we investigated the involvement of calcium in Tat endolysosome escape and subsequent LTR transactivation. We demonstrated that chelating endolysosome calcium with high-affinity rhodamine-dextran or chelating cytosolic calcium with BAPTA-AM attenuated Tat endolysosome escape and LTR transactivation. Significantly, we demonstrated that pharmacologically blocking and knocking down the endolysosome-resident two-pore channels (TPCs) attenuated Tat endolysosome escape and LTR transactivation. This calcium-mediated effect appears to be selective for TPCs because knocking down TRPML1 calcium channels was without effect. Our findings suggest that calcium released from TPCs is involved in Tat endolysosome escape and subsequent LTR transactivation. TPCs might represent a novel therapeutic target against HIV-1 infection and HIV-associated neurological complications

Two-pore channels regulate Tat endolysosome escape and Tat-mediated HIV-1 LTR transactivation.

HIV-1 Tat is essential for HIV-1 replication and appears to play an important role in the pathogenesis of HIV-associated neurological complications. Secreted from infected or transfected cells, Tat has the extraordinary ability to cross the plasma membrane. In the brain, Tat can be taken up by CNS cells via receptor-mediated endocytosis. Following endocytosis and its internalization into endolysosomes, Tat must be released in order for it to activate the HIV-1 LTR promoter and facilitate HIV-1 viral replication in the nucleus. However, the underlying mechanisms whereby Tat escapes endolysosomes remain unclear. Because Tat disrupts intracellular calcium homeostasis, we investigated the involvement of calcium in Tat endolysosome escape and subsequent LTR transactivation. We demonstrated that chelating endolysosome calcium with high-affinity rhodamine-dextran or chelating cytosolic calcium with BAPTA-AM attenuated Tat endolysosome escape and LTR transactivation. Significantly, we demonstrated that pharmacologically blocking and knocking down the endolysosome-resident two-pore channels (TPCs) attenuated Tat endolysosome escape and LTR transactivation. This calcium-mediated effect appears to be selective for TPCs because knocking down TRPML1 calcium channels was without effect. Our findings suggest that calcium released from TPCs is involved in Tat endolysosome escape and subsequent LTR transactivation. TPCs might represent a novel therapeutic target against HIV-1 infection and HIV-associated neurological complications

Incorporating steric hindrance into the additive design enables a robust formulation of alumina ink for extrusion-based 3D printing

The capabilities of additive manufacturing for fabrication of complex and thin-walled ceramic-based objects are restricted by the availability of ceramics inks. Formulations of current ink systems strictly depend on using a high content of organic additives (5-30 wt%). The high amounts of additives affect uniformity and dimensional accuracy of the final object. Here, we designed a single additive that enables printing of high aspect ratio and thin-walled structures (height/width = 58) from an ink of alumina nanoparticles that comprises very low organic content (i.e., 1.25 wt % of nanoparticles mass). In addition to the generally exploited electrostatic effect, this additive has purpose-driven tailoring to harness steric hindrance to control the viscoelastic response of ceramic suspensions and realize an optimum ink for extrusion-based 3D printing. We pursued a stepwise approach in developing such an additive through synthesis of series of copolymers with backbone monomers of 2-acrylamido-2-methylpropanesulfonic acid and acrylic acid and side chains of poly(ethylene glycol). When the optimized additive is used, the suspension attains ∼80 wt % solid loading−99% of the theoretical limit calculated by the Krieger−Dougherty equation. The shrinkage and deflection of the printed patterns as well as compactness and sinter-ability of dried structures are monitored. The printed structures did not experience any deformation or deflection during printing and reached 68% of theoretical density (TD: 3.98 g/cm3) after drying. This compactness allowed sintering at lower temperatures and improved dimensional control of the final product. Our approach to formulate ceramic inks enables the embodiment of fully aqueous systems with the utmost material content and has the potential to expand the limited portfolio of ceramic inks