Angiotensin-converting enzyme-2 (ACE2) is a critical regulator of heart function and a cellular receptor for the causative agent of severe-acute respiratory syndrome (SARS), SARS-CoV (coronavirus). ACE2 is a type I transmembrane protein, with an extracellular N-terminal domain containing the active site and a short intracellular C-terminal tail. A soluble form of ACE2, lacking its cytosolic and transmembrane domains, has been shown to block binding of the SARS-CoV spike protein to its receptor. In this study, we examined the ability of ACE2 to undergo proteolytic shedding and investigated the mechanisms responsible for this shedding event. We demonstrated that ACE2, heterologously expressed in HEK293 cells and endogenously expressed in Huh7 cells, undergoes metalloproteinase-mediated, phorbol ester-inducible ectodomain shedding. By using inhibitors with differing potency toward different members of the ADAM (a disintegrin and metalloproteinase) family of proteases, we identified ADAM17 as a candidate mediator of stimulated ACE2 shedding. Furthermore, ablation of ADAM17 expression using specific small interfering RNA duplexes reduced regulated ACE2 shedding, whereas overexpression of ADAM17 significantly increased shedding. Taken together, these data provided direct evidence for the involvement of ADAM17 in the regulated ectodomain shedding of ACE2. The identification of ADAM17 as the protease responsible for ACE2 shedding may provide new insight into the physiological roles of ACE2.

Hyperphosphorylated tau plays an important role in the formation of neurofibrillary tangles in brains of patients with Alzheimer's disease (AD) and related tauopathies and is a crucial factor in the pathogenesis of these disorders. Though diverse kinases have been implicated in tau phosphorylation, protein phosphatase 2A (PP2A) seems to be the major tau phosphatase. Using murine primary neurons from wild-type and human tau transgenic mice, we show that the antidiabetic drug metformin induces PP2A activity and reduces tau phosphorylation at PP2A-dependent epitopes in vitro and in vivo. This tau dephosphorylating potency can be blocked entirely by the PP2A inhibitors okadaic acid and fostriecin, confirming that PP2A is an important mediator of the observed effects. Surprisingly, metformin effects on PP2A activity and tau phosphorylation seem to be independent of AMPK activation, because in our experiments (i) metformin induces PP2A activity before and at lower levels than AMPK activity and (ii) the AMPK activator AICAR does not influence the phosphorylation of tau at the sites analyzed. Affinity chromatography and immunoprecipitation experiments together with PP2A activity assays indicate that metformin interferes with the association of the catalytic subunit of PP2A (PP2Ac) to the so-called MID1-α4 protein complex, which regulates the degradation of PP2Ac and thereby influences PP2A activity. In summary, our data suggest a potential beneficial role of biguanides such as metformin in the prophylaxis and/or therapy of AD.

Neurofilaments possess side arms that comprise the carboxy-terminal domains of neurofilament middle and heavy chains (NFM and NFH); that of NFH is heavily phosphorylated in axons. Here, we demonstrate that phosphorylation of NFH side arms is a mechanism for regulating transport of neurofilaments through axons. Mutants in which known NFH phosphorylation sites were mutated to preclude phosphorylation or mimic permanent phosphorylation display altered rates of transport in a bulk transport assay. Similarly, application of roscovitine, an inhibitor of the NFH side arm kinase Cdk5/p35, accelerates neurofilament transport. Analyses of neurofilament movement in transfected living neurons demonstrated that a mutant mimicking permanent phosphorylation spent a higher proportion of time pausing than one that could not be phosphorylated. Thus, phosphorylation of NFH slows neurofilament transport, and this is due to increased pausing in neurofilament movement.

Neurofilaments are transported through axons by slow axonal transport. Abnormal accumulations of neurofilaments are seen in several neurodegenerative diseases, and this suggests that neurofilament transport is defective. Excitotoxic mechanisms involving glutamate are believed to be part of the pathogenic process in some neurodegenerative diseases, but there is currently little evidence to link glutamate with neurofilament transport. We have used a novel technique involving transfection of the green fluorescent protein–tagged neurofilament middle chain to measure neurofilament transport in cultured neurons. Treatment of the cells with glutamate induces a slowing of neurofilament transport. Phosphorylation of the side-arm domains of neurofilaments has been associated with a slowing of neurofilament transport, and we show that glutamate causes increased phosphorylation of these domains in cell bodies. We also show that glutamate activates members of the mitogen-activated protein kinase family, and that these kinases will phosphorylate neurofilament side-arm domains. These results provide a molecular framework to link glutamate excitotoxicity with neurofilament accumulation seen in some neurodegenerative diseases.

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A number of muscular dystrophies are associated with the defective glycosylation of alpha-dystroglycan and many are now known to result from mutations in a number of genes encoding putative or known glycosyltransferases. These diseases include severe forms of congenital muscular dystrophy (CMD) such as Fukuyama type congenital muscular dystrophy (FCMD), Muscle-Eye-Brain disease (MEB) and Walker-Warburg syndrome (WWS), which are associated with brain and eye abnormalities. The defective glycosylation of alpha-dystroglycan in these disorders leads to a failure of alpha-dystroglycan to bind to extra-cellular matrix components and previous attempts to model these disorders have shown that the generation of fukutin- and Pomt1-deficient knockout mice results in early embryonic lethality due to basement membrane defects. We have used the zebrafish as an animal model to investigate the pathological consequences of downregulating the expression of the putative glycosyltransferase gene fukutin-related protein (FKRP) on embryonic development. We have found that downregulating FKRP in the zebrafish results in embryos which develop a range of abnormalities reminiscent of the developmental defects observed in human muscular dystrophies associated with mutations in FKRP. FKRP morphant embryos showed a spectrum of phenotypic severity involving alterations in somitic structure and muscle fibre organization as well as defects in developing neuronal structures and eye morphology. The pathological phenotype was found to correlate with a reduction in alpha-dystroglycan glycosylation and reduced laminin binding. Further characterization of the developmental processes affected in FKRP morphant embryos may lead to a better understanding of the pathological spectrum observed in muscular dystrophies associated with mutations in the human FKRP gene.

Neurofilaments are among the most abundant organelles in neurones. They are synthesised in cell bodies and then transported into and through axons by a process termed 'slow axonal transport' at a rate that is distinct from that driven by conventional fast motors. Several recent studies have now demonstrated that this slow rate of transport is actually the consequence of conventional fast rates of movement that are interrupted by extended pausing. At any one time, most neurofilaments are thus stationary. Accumulations of neurofilaments are a pathological feature of several human neurodegenerative diseases suggesting that neurofilament transport is disrupted in disease states. Here, we review recent advances in our understanding of neurofilament transport in both normal and disease states. Increasing evidence suggests that phosphorylation of neurofilaments is a mechanism for regulating their transport properties, possibly by promoting their detachment from the motor(s). In some neurodegenerative diseases, signal transduction mechanisms involving neurofilament kinases and phosphatases may be perturbed leading to disruption of transport.

Collapsin response mediator protein-2 (CRMP2) was recently identified as a physiological substrate for GSK3 and Cdk5, two protein kinases suggested to exhibit greater activity in Alzheimer's disease (AD). Indeed, phosphorylation of CRMP2, at the residues targeted by GSK3 and Cdk5, is relatively high in cortex isolated from human AD brain, as well as in the brains of animal models of AD, while phospho-CRMP2 is found in neurofibrillary tangles. In mouse models of AD, increased phosphorylation occurs prior to pathology. Although CRMP2 has no known enzymatic activity, a great deal of information is appearing on its importance in neuronal development and polarity, as well as in axon growth and guidance. In this mini-review, we examine what is known about CRMP2 function, how that is controlled by phosphorylation, what alterations in molecular mechanisms could lead to the abnormally high CRMP2 phosphorylation in AD, and whether this is likely to be specific to AD or occur in other forms of neurodegeneration. This will include discussion of the evidence for increased GSK3 or Cdk5 activity, for decreased phosphatase activity, or the upregulation of other CRMP2 protein kinases in AD. Importantly, we will compare the processes that may contribute to increased CRMP2 phosphorylation with those known to increase tau hyperphosphorylation in AD, and whether these are likely to be part of disease development or a useful early marker for AD.