Role of calcium in inflammation: Relevance to Alzheimer's disease

Alzheimer’s disease (AD) is neuropathologically characterized by excessive beta -amyloid (Aβ) plaques and neurofibrillary tangles composed of hyperphosphorylated tau in the brain. Although the etiology of genetic cases of AD has been attributed to mutations in presenilin and amyloid precursor protein (APP) genes, in most sporadic cases of AD, the etiology is still unknown and various predisposing factors could contribute to the pathology of AD. Predominant among these possible predisposing factors that have been implicated in AD are age, hypertension, traumatic brain injury, diabetes, chronic neuroinflammation, alteration in calcium levels and oxidative stress. Since both inflammation and altered calcium levels are implicated in the pathogenesis of AD, we wanted to study the effect of altered levels of calcium on inflammation and the subsequent effect of selective calcium channel blockers on the production of pro-inflammatory cytokines and chemokines. Our hypothesis is that Aβ, depending on it conformation, may contribute to altered levels of intracellular calcium in neurons and glial cells. We wanted to determine which conformation of Aβ was most pathogenic in terms of increasing inflammation and calcium influx and further elucidate the possibility of a link between altered calcium levels and inflammation. In addition, we wanted to test whether calcium channel blockers could inhibit the inflammation mediated by the most pathogenic form of Aβ, by antagonizing the calcium influx triggered by Aβ. Our results in human glial and neuronal cells demonstrate that the high molecular weight oligomers are the most potent at stimulating the release of pro-inflammatory cytokines IL-6 and IL-8 as well as increasing intracellular levels of calcium compared to other conformations of Aβ. Further, L-type calcium channel blockers and calmodulin kinase inhibitors are able to significantly reduce the levels of IL-6 and IL-8. These results suggest that Aβ-induced alteration of intracellular calcium levels contributes to its pro-inflammatory effect.

Selective and specific activation of Rab5 during endocytosis of receptor tyrosine kinases

The Rab family of proteins are low molecular weight GTPases that have the ability to switch between GTP- (active) and GDP- (inactive) bound form, and in that sense act as molecular switches. Through distinct localization on various vesicles and organelles and by cycling through GTP/GDP bound forms, Rabs are able to recruit and activate numerous effector proteins, both spatially and temporally, and hence behave as key regulators of trafficking in both endocytic and biosynhtetic pathways. The Rab5 protein has been shown to regulate transport from plasma membrane to the early endosome as well as activate signaling pathways from the early endosome. This dissertation focused on understanding Rab5 activation via endocytosis of receptor tyrosine kinases (RTKs). First, tyrosine kinase activity of RTKs was linked to endosome fusion by demonstrating that tyrosine kinase inhibitors block endosome fusion and activation of Rab5, and a constitutively active form of Rab5 is able to rescue endosome fusion. However, depending on how much ligand is available at the cell surface, the receptor-ligand complexes can be internalized via a number of distinct pathways. Similarly, Rab5 was activated in a ligand-dependent concentration dependent manner via clathrin- and caveolin-mediated pathways, as well as a pathway independent of both. However, overexpression Rabex-5, a nucleotide exchange factor for Rab5, is able to rescue activation even when all of the pathways of EGF-receptor internalization were blocked. Next, the three naturally occurring splice variants of Rabex-5 selectively activated Rab5. Lastly, Rabex-5 inhibits differentiation of 3T3-L1 and PC12 cells through 1) degradation of signaling endosome via Rab5-dependent fusion with the early endosome, 2) and inhibition of signaling cascade via ubiquitination of Ras through the ZnF domain at the N-terminus of Rabex-5. In conclusion, these data shed light on complexity of the endosomal trafficking system where tyrosine kinase activity of the receptor is able to affect endosome fusion; how different endocytic pathways affect activation of one of the key regulators of early endocytic events; and how selective activation of Rab5 via Rabex-5 can control adipogenesis and neurogenesis.

Selective and Specific Activation of Rab5 during Endocytosis of Receptor Tyrosine Kinases

The Rab family of proteins are low molecular weight GTPases that have the ability to switch between GTP- (active) and GDP- (inactive) bound form, and in that sense act as molecular switches. Through distinct localization on various vesicles and organelles and by cycling through GTP/GDP bound forms, Rabs are able to recruit and activate numerous effector proteins, both spatially and temporally, and hence behave as key regulators of trafficking in both endocytic and biosynhtetic pathways. The Rab5 protein has been shown to regulate transport from plasma membrane to the early endosome as well as activate signaling pathways from the early endosome. This dissertation focused on understanding Rab5 activation via endocytosis of receptor tyrosine kinases (RTKs). First, tyrosine kinase activity of RTKs was linked to endosome fusion by demonstrating that tyrosine kinase inhibitors block endosome fusion and activation of Rab5, and a constitutively active form of Rab5 is able to rescue endosome fusion. However, depending on how much ligand is available at the cell surface, the receptor-ligand complexes can be internalized via a number of distinct pathways. Similarly, Rab5 was activated in a ligand-dependent concentration dependent manner via clathrin- and caveolin-mediated pathways, as well as a pathway independent of both. However, overexpression Rabex-5, a nucleotide exchange factor for Rab5, is able to rescue activation even when all of the pathways of EGF-receptor internalization were blocked. Next, the three naturally occurring splice variants of Rabex-5 selectively activated Rab5. Lastly, Rabex-5 inhibits differentiation of 3T3-L1 and PC12 cells through 1) degradation of signaling endosome via Rab5-dependent fusion with the early endosome, 2) and inhibition of signaling cascade via ubiquitination of Ras through the ZnF domain at the N-terminus of Rabex-5. In conclusion, these data shed light on complexity of the endosomal trafficking system where tyrosine kinase activity of the receptor is able to affect endosome fusion; how different endocytic pathways affect activation of one of the key regulators of early endocytic events; and how selective activation of Rab5 via Rabex-5 can control adipogenesis and neurogenesis.