Yeast Reveal a "Druggable" Rsp5/Nedd4 Network that Ameliorates a-Synuclein Toxicity in Neurons

As we have mentioned before, there are currently no therapies for PD which target the underlying neurodegenerative process. Recent research published in Science, from Susan Lindquist's group (MIT), reveals pathways which could form ideal drug targets for neuroprotective treatments for PD.

Tardiff et al. used yeast cells expressing pathogenic α-synuclein (the main disease causing protein in PD) as a model for the cellular pathology in PD. Although these cells are not neurons, the α-synuclein causes very similar intracellular problems to that those found in the neurons of patients with PD and the inhibition of growth of the yeast cells provides a simple end-point for testing potential treatments.

Using this model, the authors proved that the compound N-aryl benzimidazole (NAB) strongly protects cells from α-synuclein toxicity and identified a network of proteins through which NAB exerts its protective effects. At the centre of this network was the protein Rsp5/Nedd4 which is involved in intracellular transport. The authors then showed that α-synuclein impairs intracellular transport and that NAB rescues Rsp5/Nedd4-dependent intracellular transport processes.

Thus, this study not only identifies Rsp5/Nedd4 as a new potential target for neuroprotective treatments, but also reveals the importance of dysregulated intracellular trnaport, which may drive the other pathological processes leading to neurodegeneration in PD.

How was this compound, NAB, initially identified?

Rather than selectively targeting cellular processes known to be involved in neurodegenerative diseases, the authors ran an 'unbiased' screen, more-or-less randomly testing over 190,000 different compounds for their ability to restore the growth of yeast cells following treatment with TDP-43 (another protein implicated in neurodegenerative diseases) (Tardiff et al, 2012).

 

NAB was initially shown to have protective effects in a yeast model expressing TDP-43 (Tardiff et al, 2012) but the group subsequently found it to be even more beneficial in a model expressing AS (a model of PD)

Of course this is not very efficient, but the beauty of the yeast model is that it is very cheap and quick to run, so such huge numbers of compounds can be easily tested.

By employing this 'unbiased' approach the authors were able to identify completely new therapeutic targets, rather than being restricted to potential targets based on our (currently quite poor) knoweldge of the cellular mechanisms involved in neurodegeneration.

How was NAB shown to have protective effects in models of PD?

In the yeast model of PD, NAB  prevented the accumulation of vesicular α-synuclein foci, the generation of reactive oxygen species, the block in ER-golgi trafficking and the nitration of proteins; all of which are part of the neurodegerative process in PD.

The authors then confirmed that NAB could also rescue neurons from the effects of α-synuclein in a nematode and a rat model of PD and finally in cells taken from PD patients which were turned into stem cells (iPSCs) and then differentiated into neurons.

Interestingly, NAB had no effect on levels of α-synuclein within the cell, indicating that it worked by inhibiting the down-stream damaging effects of α-synuclein, rather than preventing the accumulation of α-synucleinitself.

What are the mechanisms through which NAB has a protective effect?

A genetic screen of mutations in thousands of different genes then identified a core network of proteins through which NAB has its effects:

 

The network of genes with which NAD interacts, as indentified through a mutation screen. Colours represent type of mutation (green = overexpression, red = SNP, blue = transposon insertion, yellow = knock-out)

At the centre of this network is the protein Rsp5 (which is called Nadd4 in mammals). Rsp5 is a 'ubiquitin ligase' which promotes endosomal transport. NAB was found to exert many of its protective effects through this central node of the network. Specifically, NAB was found to promote the Rsp5-dependent endocytosis and delivery to the vacuole of the protein Mup1 and to promote the Rsp5-dependent Golgi-to-vacuole trafficking of the protein Sna3.

In the yeast model of PD, expression of AS was found to impair endosomal transport from the plasma membrane to the vacuole, as well as trafficking from the Golgi body to vacuoles; both these processes were rescued by NAB.

Why is this work really exciting?

NAB is a very long way from clinical trials -- it has yet to be tested in animal models, let alone humans -- thus, the protective effects of this compound are, on their own, not such exciting findings.

 Rather, the most interesting finding from this study is the revelation of the importance of Rsp5/Nedd4 in endosomal and ER-Golgi transport in PD: processes which are only recently becoming recognised as key to the underlying pathological process in PD (Esposito et al, 2012).

 

Dysfunctional endosomal and ER-Golgi trafficking contribute to core PD pathology and NAB restores these protective processes via Rsp5.

Tardiff et al. speculate that the processes we often consider to be the causes of neurodegeneration in PD (such as ROS production and mitochondrial dysfunction) are in fact secondary to the core pathology of intracellular transport dysfunction. Thus, in order to curb neurodegeneration in PD, it may be more fruitful to target these direct effects of AS, rather than the downstream secondary pathologies. Either way, dysfunctional intracellular trafficking in PD is line of research which warrants further attention.

References:



Lindquist S. (2013). Yeast Reveal a "Druggable" Rsp5/Nedd4 Network that Ameliorates a-Synuclein Toxicity in Neurons Science DOI: 10.1126/science.1245321

 
D.F. Tardiff, M. L. Tucci, K. A. Caldwell, G.A. Caldwell, S. Lindquist, Different 8-hydroxyquinolines protect models of TDP-43 protein, a-synuclein and polyglutamine proteotoxicity through distinct mechanisms. J. Biol. Chem. 287, 4107-4120 (2012).


G. Esposito, F. Ana Clara, P. Verstreken, Synaptic vesicle trafficking and Parkinson's disease. Dev. Neurobiol. 72, 134-144 (2012). Tardiff DF, Jui NT, Khurana V, Tambe MA, Thompson ML, Chung CY, Kamadurai HB, Kim HT, Lancaster AK, Caldwell KA, Caldwell GA, Rochet JC, Buchwald SL,