PI31 expression prevents neuronal degeneration in a mouse Parkinson Disease model
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Abstract
Age-related neurodegenerative diseases pose a major unmet health need since no effective treatment strategies are currently available. These disorders are defined by the accumulation of abnormal protein aggregates that impair synaptic function and cause progressive neuronal degeneration. Therefore, stimulating protein clearance mechanisms may be neuro-protective. The proteasome regulator PI31 promotes local protein degradation at synapses by mediating fast proteasome transport in neurites, and loss of PI31 function causes neuronal degeneration. Here we show that transgenic expression of PI31 in a mouse Parkinson’s Disease model preserves neuronal function and greatly extends animal health and lifespan. These results indicate that targeting the PI31-pathway may have therapeutic value for treating neurodegenerative disorders.
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###Reviewer #2:
In 2011 these authors showed that Drosophila DmPI31 is a binding partner of the F box protein Nutcracker, a component of an SCF ubiquitin ligase (E3) required for caspase activation during sperm differentiation in Drosophila. DmPI31 binds Nutcracker via a mechanism that is also used by mammalian FBXO7 and PI31. Subsequently, they have shown that PI31 serves as an adaptor to couple proteasomes with dynein light chains and inactivation of PI31 inhibited proteasome motility in axons and disrupted synaptic proteostasis, structure, and function. In addition, conditional loss of PI31 in spinal motor neurons (MNs) and cerebellar Purkinje cells (PCs) caused axon degeneration, neuronal loss, and progressive spinal and cerebellar neurological dysfunction.
Here the authors show that like Fbxo7 mutant mice, PI31 conditional KO mice …
###Reviewer #2:
In 2011 these authors showed that Drosophila DmPI31 is a binding partner of the F box protein Nutcracker, a component of an SCF ubiquitin ligase (E3) required for caspase activation during sperm differentiation in Drosophila. DmPI31 binds Nutcracker via a mechanism that is also used by mammalian FBXO7 and PI31. Subsequently, they have shown that PI31 serves as an adaptor to couple proteasomes with dynein light chains and inactivation of PI31 inhibited proteasome motility in axons and disrupted synaptic proteostasis, structure, and function. In addition, conditional loss of PI31 in spinal motor neurons (MNs) and cerebellar Purkinje cells (PCs) caused axon degeneration, neuronal loss, and progressive spinal and cerebellar neurological dysfunction.
Here the authors show that like Fbxo7 mutant mice, PI31 conditional KO mice have a decreased testis and thymus size and motor neuron specific loss of either FBXO7 or PI31 produced similar phenotypes in motor neurons. They generated a mouse that conditionally expressed FLAG-tagged PI31 this could rescue PI31 mutant mice; this transgene (under a Chat driver) rescued the phenotype of FBXO7 mutant mice from which they concluded that the consequences of FBXO7 mutation relate to loss of PI31 function in the cell types studied.
FBXO7 is the substrate recognition module of a novel proteasome‐interacting E3 ubiquitin ligase. In addition to binding PI31, FBXO7 also drives PI31 ubiquitylation and thus regulates its cellular levels. That the transgene can rescue the phenotype in the Chat-expressing cells is surprising and striking. However, it would necessary to reveal more about the underlying molecular mechanism. In the cell types rescued, is there another E3 ligase with overlapping substrate specificity? Are there mitochondrial phenotypes that are not rescued?
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###Reviewer #1:
This manuscript focuses on the role played by the PI31 protein in regulating presynaptic proteasome abundance and the health of motor neurons. In particular, it presents striking data from knockout and conditional KO mice showing that depletion of PI31 and Fbxo7/PARK15 (Parknson's disease gene) yield similar phenotypes, including motor neuron defects, following their conditional depletion. Furthermore, in the absence of Fbxo7/PARK15, PI31 levels were greatly reduced. This suggested that a major role for Fbxo7 is to promote the abundance/stability of PI31. In support of this model, transgenic expression of PI31 completely rescued overall health, body weight and motor neuron morphology in Fbxo7 mutant mice. These results are impressive. However, the manuscript implies but does not show that the mechanism through which PI31 …
###Reviewer #1:
This manuscript focuses on the role played by the PI31 protein in regulating presynaptic proteasome abundance and the health of motor neurons. In particular, it presents striking data from knockout and conditional KO mice showing that depletion of PI31 and Fbxo7/PARK15 (Parknson's disease gene) yield similar phenotypes, including motor neuron defects, following their conditional depletion. Furthermore, in the absence of Fbxo7/PARK15, PI31 levels were greatly reduced. This suggested that a major role for Fbxo7 is to promote the abundance/stability of PI31. In support of this model, transgenic expression of PI31 completely rescued overall health, body weight and motor neuron morphology in Fbxo7 mutant mice. These results are impressive. However, the manuscript implies but does not show that the mechanism through which PI31 supports neuronal health is by promoting the axonal transport of proteasomes and thus suppressing the presynaptic accumulation of ubiquitinated proteins. Several key experiments to address this issue would greatly strengthen the manuscript (outlined below).
- Major statements are made about the importance of PI31 for axonal transport of proteasomes and presynaptic aggregate clearance. In order to establish that PI31 is indeed supporting neuronal health by promoting axonal transport of proteasomes and clearing presynaptic protein aggregates, it is necessary to show:
-- That motor neuron presynaptic proteasome number is reduced in the PI31 and Fbxo7 KO mice and restored in the Fbxo7 mutant mice that express the PI31 transgene.
-- That expression of the PI31 transgene in the Fbxo7 mutant mice suppresses the presynaptic accumulation of P62 aggregates.
It would be helpful if the abstract defined the Parkinson's disease model (PARK15) that was investigated.
Quantification of the presynaptic P62 aggregate phenotype in figure 2 would be helpful as would including a higher magnification image of the wildtype synapse with the P62 labeling.
Given that the major phenotypes that are characterized are not directly related to Parkinson's disease, the upfront emphasis on Parkinson's disease might not be warranted. Although the mouse phenotypes that are reported are striking, the title in particular suggests a more direct connection to this disease than is warranted by the data.
Figures 3C and 4B: Individual data points should be plotted and a statistical test would be helpful.
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##Preprint Review
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###Summary:
There is great interest in understanding the molecular basis of FBXO7/PARK15 pathogenesis and the present, high quality story includes an impressive rescue in cells transgenically overexpressing PI31 protein. Nevertheless, as discussed in greater detail below, the two reviewers felt that more work would be needed to document the molecular basis for this phenotype rescue.
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