Richard Tuxworth

Interactions between the juvenile Batten disease gene, CLN3, and the Notch and JNK signalling pathways

Mutations in the gene CLN3 are responsible for the neurodegenerative disorder juvenile neuronal ceroid lipofuscinosis or Batten disease. CLN3 encodes a multi-spanning and hydrophobic transmembrane protein whose function is unclear. As a consequence, the cell biology that underlies the pathology of the disease is not well understood. We have developed a genetic gain-of-function system in Drosophila to identify functional pathways and interactions for CLN3. We have identified previously unknown interactions between CLN3 and the Notch and Jun N-terminal kinase signalling pathways and have uncovered a potential role for the RNA splicing and localization machinery in regulating CLN3 function.

The Batten disease gene CLN3 is required for the response to oxidative stress

Mutations in the CLN3 gene cause juvenile neuronal ceroid lipofuscinosis (JNCL or Batten disease), an early onset neurodegenerative disorder. JNCL is the most common of the NCLs, a group of disorders with infant or childhood onset that are caused by single gene mutations. The NCLs, although relatively rare, share many pathological and clinical similarities with the more common late-onset neurodegenerative disorders, while their simple genetic basis makes them an excellent paradigm. The early onset and rapid disease progression in the NCLs suggests that one or more key cellular processes are severely compromised. To identify the functional pathways compromised in JNCL, we have performed a gain-of-function modifier screen in Drosophila. We find that CLN3 interacts genetically with the core stress signalling pathways and components of stress granules, suggesting a function in stress responses. In support of this, we find that Drosophila lacking CLN3 function are hypersensitive to oxidative stress yet they respond normally to other physiological stresses. Overexpression of CLN3 is sufficient to confer increased resistance to oxidative stress. We find that CLN3 mutant flies perceive conditions of increased oxidative stress correctly but are unable to detoxify reactive oxygen species, suggesting that their ability to respond is compromised. Together, our data suggest that the lack of CLN3 function leads to a failure to manage the response to oxidative stress and this may be the key deficit in JNCL that leads to neuronal degeneration.

Asymmetric localisation of Miranda and its cargo proteins during neuroblast division requires the anaphase-promoting complex/cyclosome.

Asymmetric cell divisions generate cell fate diversity during both invertebrate and vertebrate development. Drosophila neural progenitors or neuroblasts (NBs) each divide asymmetrically to produce a larger neuroblast and a smaller ganglion mother cell (GMC). The asymmetric localisation of neural cell fate determinants and their adapter proteins to the neuroblast cortex during mitosis facilitates their preferential segregation to the GMC upon cytokinesis. In this study we report a novel role for the anaphase-promoting complex/cyclosome (APC/C) during this process. Attenuation of APC/C activity disrupts the asymmetric localisation of the adapter protein Miranda and its associated cargo proteins Staufen, Prospero and Brat, but not other components of the asymmetric division machinery. We demonstrate that Miranda is ubiquitylated via its C-terminal domain; removal of this domain disrupts Miranda localisation and replacement of this domain with a ubiquitin moiety restores normal asymmetric Miranda localisation. Our results demonstrate that APC/C activity and ubiquitylation of Miranda are required for the asymmetric localisation of Miranda and its cargo proteins to the NB cortex.

Modification of the Drosophila model of in vivo Tau toxicity reveals protective phosphorylation by GSK3β.

Summary Hyperphosphorylation of the microtubule associated protein, Tau, is the hallmark of a group of neurodegenerative disorders known as the tauopathies which includes Alzheimers disease. Precisely how and why Tau phosphorylation is increased in disease is not fully understood, nor how individual sites modify Tau function. Several groups have used the Drosophila visual system as an in vivo model to examine how the toxicity of Tau varies with phosphorylation status. This system relies on overexpression of Tau from transgenes but is susceptible to position effects altering expression and activity of the transgenes. We have refined the system by eliminating position effects through the use of site-specific integration. By standardising Tau expression levels we have been able to compare directly the toxicity of different isoforms of Tau and Tau point mutants that abolish important phosphorylation events. We have also examined the importance of human kinases in modulating Tau toxicity in vivo. We were able to confirm that human GSK3&bgr; phosphorylates Tau and increases toxicity but, unexpectedly, we identified that preventing phosphorylation of Ser404 is a protective event. When phosphorylation at this site is prevented, Tau toxicity in the Drosophila visual system is increased in the presence of GSK3&bgr;. Our data suggest that not all phosphorylation events on Tau are associated with toxicity.

in vivo localization of the neuronal ceroid lipofuscinosis proteins, CLN3 and CLN7, at endogenous expression levels

The neuronal ceroid lipofuscinoses are a group of recessively inherited, childhood-onset neurodegenerative conditions. Several forms are caused by mutations in genes encoding putative lysosomal membrane proteins. Studies of the cell biology underpinning these disorders are hampered by the poor antigenicity of the membrane proteins, which makes visualization of the endogenous proteins difficult. We have used Drosophila to generate knock-in YFP-fusions for two of the NCL membrane proteins: CLN7 and CLN3. The YFP-fusions are expressed at endogenous levels and the proteins can be visualized live without the need for overexpression. Unexpectedly, both CLN7 and CLN3 have restricted expression in the CNS of Drosophila larva and are predominantly expressed in the glia that form the insect blood-brain-barrier. CLN7 is also expressed in neurons in the developing visual system. Analogous with murine CLN3, Drosophila CLN3 is strongly expressed in the excretory and osmoregulatory Malpighian tubules, but the knock-in also reveals unexpected localization of the protein to the apical domain adjacent to the lumen. In addition, some CLN3 protein in the tubules is localized within mitochondria. Our in vivo imaging of CLN7 and CLN3 suggests new possibilities for function and promotes new ideas about the cell biology of the NCLs.

Asymmetric Cell Division Miranda Chauffeured by Jaguar

Drosophila neuroblasts divide to produce daughters of distinct fate by asymmetrically localizing cell fate determinants and segregating them preferentially to one daughter. An intact F-actin cytoskeleton was known to be required, and now a myosin VI (Jaguar) has been shown to be necessary for basal targeting of cell fate determinants in neuroblasts.