The programmes of work in the laboratory are currently aimed at understanding the molecular mechanisms and physiological significance of intracellular signalling networks which involve a family of enzymes called phosphoinositide 3OH-kinases (PI3Ks).
PI3Ks are now accepted to be critical regulators of numerous important and complex cell responses, including cell growth, division, survival and movement.
PI3Ks catalyse the formation of one or more critical phospholipid messenger molecules, which signal information by binding to specific domains in target proteins. Currently the best understood pathway involves the activation of Class I PI3Ks by cell surface receptors.
In recent years, the Hawkins laboratory has increasingly focused on the role of PI3Ks in the signalling mechanisms which allow receptors on neutrophils (white blood cells) to control various aspects of neutrophil function.
Neutrophils are key players in the front line of our immune system, responsible primarily for the recognition and destruction of bacterial and fungal pathogens. However, they are also involved in the amplification cascades that underlie various inflammatory pathologies, e.g. Acute Respiratory Distress Syndrome (ARDS) and rheumatoid arthritis.
Abnormalities in the endosomal-autophagic-lysosomal (EAL) system are an early event in Alzheimer's disease (AD) pathogenesis. However, the mechanisms underlying these abnormalities are unclear. The transient receptor potential channel mucolipin 1(TRPML1), a vital endosomal-lysosomal Ca2+ channel whose loss of function leads to neurodegeneration, has not been investigated with respect to EAL pathogenesis in late onset AD (LOAD). Here, we identify pathological hallmarks of TRPML1 dysregulation in LOAD neurons, including increased perinuclear clustering and vacuolation of endolysosomes. We reveal that iPSC human cortical neurons expressing APOE e4, the strongest genetic risk factor for LOAD, have significantly diminished TRPML1-induced endolysosomal Ca2+ release. Furthermore, we found that blocking TRPML1 function in primary neurons by depleting the TRPML1 agonist PI(3,5)P2. via PIKfyve inhibition, recreated multiple features of EAL neuropathology evident in LOAD. This included increased endolysosomal Ca2+ content, enlargement and perinuclear clustering of endolysosomes, autophagic vesicle accumulation, and early endosomal enlargement. Strikingly, these AD-like neuronal EAL defects were rescued by TRPML1 reactivation using its synthetic agonist ML-SA1. These findings implicate defects in TRPML1 in LOAD EAL pathogenesis and present TRPML1 as a potential therapeutic target.
Host defense against bacterial and fungal infections diminishes with age. In humans, impaired neutrophil responses are thought to contribute to this decline. However, it remains unclear whether neutrophil responses are also impaired in old mice. Here, we investigated neutrophil function in old mice, focusing on responses primed by lipopolysaccharide (LPS), an endotoxin released by gram-negative bacteria like , which signals through toll-like receptor (TLR) 4. We show that old mice have a reduced capacity to clear pathogenic during septic peritonitis. Neutrophil recruitment was elevated during LPS-induced but not aseptic peritonitis. Neutrophils from old mice showed reduced killing of . Their reactive oxygen species (ROS) production was impaired upon priming with LPS but not with GM-CSF/TNFα. Phagocytosis and degranulation were reduced in a partially LPS-dependent manner, whereas impairment of NET release in response to was independent of LPS. Unexpectedly, chemotaxis was normal, as were Rac1 and Rac2 GTPase activities. LPS-primed activation of Erk and p38 Mapk was defective. PIP production was reduced upon priming with LPS but not with GM-CSF/TNFα, whereas PIP levels were constitutively low. The expression of 5% of neutrophil proteins was dysregulated in old age. Granule proteins, particularly cathepsins and serpins, as well as TLR-pathway proteins and membrane receptors were upregulated, whereas chromatin and RNA regulators were downregulated. The upregulation of CD180 and downregulation of MyD88 likely contribute to the impaired LPS signaling. In summary, all major neutrophil responses except chemotaxis decline with age in mice, particularly upon LPS priming. This LPS/TLR4 pathway dependence resolves previous controversy regarding effects of age on murine neutrophils and confirms that mice are an appropriate model for the decline in human neutrophil function.
Li et al present the results of a proximity-interaction screen in mammalian cells for the effector proteins of 25 members of the Arf family of small GTPases. This study has generated an important resource for those working in several areas of cell biology and provided an initial characterisation of two new cellular roles for some of the least well studied members of this family, the regulation of PLD1 by ARL11/14 in phagocytosis, and the regulation of PI4KB by ARL5A/5B in the Golgi.