regeneration of injured or unhealthy axons holds great therapeutic promise for neurological disorders including acute trauma to the brain or spinal cord , stroke , and neurodegenerative diseases .
the phosphatase and tensin homolog ( pten ) gene has emerged as an important regulator of axon regeneration , and recent findings by multiple groups support the potential of using pten as a therapeutic target .
our recent paper reports that long - term deletion of pten , a tumor suppressor gene , does not result in any major detectable pathology and may also enhance neuronal vitality ( gutilla et al . , 2016 ) . by selectively deleting pten in the motor cortex of young mice , we specifically assessed the effects of pten loss using the same approach that has been used to promote regeneration of the corticospinal tract after spinal cord injury .
pten 's impact on axon regeneration was discovered in a seminal experiment in which conditional genetic deletion of pten promoted robust axonal regeneration of retinal ganglion cells ( rgcs ) following optic nerve crush ( park et al . , 2008 ) .
the rationale for this experimental approach is rooted in the discovery of inhibitory intrinsic and extrinsic factors that prevent regeneration of injured axons ( schwab and bartholdi , 1996 ; fitch and silver , 2008 ) .
one approach to overcoming the intrinsic inhibitory factors focuses on attempting to  recapitulate development  , in order to return adult neurons to a more growth permissive state ( filbin , 2006 ) .
growth cessation after the completion of development is thought to occur in part due to the onset of growth inhibiting genes being expressed . in their landmark study , park et al .
( 2008 ) tested whether axon regeneration could be enhanced if genes that normally repress cell growth were eliminated prior to axon injury .
they specifically examined several known tumor suppressor genes including pten , p53 , retinoblastoma , smad4 , dicer , and lkb .
the effect of each individual gene was studied using multiple strains of  floxed  mice , with each strain having only one of the aforementioned genes flanked by lox - p sites .
the specific gene was deleted in the retina be injecting aav - cre into the vitreous humor of the eye prior to performing an optic nerve crush .
only deletion of the pten gene enabled axotomized rgcs to regenerate , though deletion of both pten and p53 reduced retrograde degeneration of rgcs that otherwise occurred .
the latter finding , not emphasized at the time , could mean that deleting pten enhanced rgc vitality such that the neurons could survive traumatic injuries that would normally cause cell death .
following the initial finding that linked pten deletion to enhanced neural regeneration , a follow up study tested whether neuronal pten deletion could also enhance regeneration after spinal cord injury .
damage to cst axons due to spinal cord injury is the cause of paralysis , and enabling regeneration of the cst is the best hope for restoring motor function after injury .
( 2008 ) , this study used floxed pten mice , and pten was deleted in the motor cortex of mice one day after birth by injecting aav - cre into the sensorimotor cortex .
tract tracing of cst axons revealed robust and unprecedented regeneration beyond the injury site ( liu et al . , 2010 ) .
. genetic deletion of pten soon after spinal cord injury in adult mice , or knockdown of pten expression using short hairpin rna ( shrna ) against pten in adult rats a few days before injury was found to enhance the regenerative growth of the adult cst and recovery of skilled motor functions ( zukor et al .
, 2013 ; lewandowski and steward , 2014 ; danilov and steward , 2015 ) .
remarkably , pten deletion also induced robust cst regeneration in the chronic injury setting one year following injury ( du et al . , 2015 ) .
these discoveries further highlight the potential of using pten interference as a pro - regenerative strategy for treating adult spinal cord injury ( ramon - cueto et al . , 2000 ) .
the pten gene is thought to exert its growth inhibiting effects through the pten protein 's negative regulation of phosphoinositide 3-kinase ( pi3k ) . as a phosphatase
, pten converts active phosphatidylinositol ( 3 , 4,5)-triphosphate ( pip3 ) to inactive phosphatidylinositol ( 4,5)-bisphosphate ( pip2 ) , resulting in diminished akt and downstream mammalian target of rapamycin ( mtor ) activation .
thus , deletion of pten leads to enhanced levels of pip3 , activation of akt , and activation of mtor .
the mtor pathway is well known for its ability to regulate cell growth and proliferation and pten 's upstream and non - redundant negative regulation of the mtor pathway make it a promising pro - regenerative therapeutic target ( don et al . , 2012 ) .
it 's here that we come to the theme of our review ; is it possible to take the brakes off of such a powerful growth - promoting pathway without losing control ?
the logic behind testing pten was that it had been identified as a tumor suppressor gene .
pten mutations are common in several cancers , and have been associated with developmental disorders including macrocephaly and autism spectrum disorders ( goffin et al . , 2001 ; hollander et al . ,
experimental studies in which pten was deleted during early development in particular cell types revealed neuronal over - growth , brain enlargement , seizures , and premature death .
these studies used mice with a lox - p flanked pten gene paired with cre recombinase expression regulated under the control of promoters including neuron specific enolase ( nse , ( kwon et al . , 2006 ) ) , glial fibrillary acid protein ( gfap , ( backman et al . , 2001 ; kwon et al .
yue et al . , 2005 ; fraser et al . , 2008 ; wen et al . , 2013
) ) , ca / calmodulin - dependent protein kinase ii ( camkii , ( sperow et al . , 2012 ) ) , and the dopamine active transporter ( dat , ( diaz - ruiz et al . , 2009 ) ) . in studies using nse , gfap , and camkii promoter driven cre expression , mice with pten deletion exhibit significantly higher postnatal mortality and premature death ( ~11 weeks of age for camkii - cre , ( sperow et al . , 2012 ) ) . in the gfap - cre models ,
multiple groups have identified neurons with successful pten deletion in the cerebellum , hippocampus , and the cerebral cortex ( backman et al . , 2001 ; kwon et al . , 2001 ; fraser et al .
the negative consequences following widespread neuronal pten loss during development necessitated an in - depth examination of the long - term consequences of deleting pten in the way that promotes axon regeneration . as a first step in assessing the potential risk
, we employed the same experimental model as in our original report of cst regeneration following spinal cord injury ( liu et al . , 2010 ) .
pten was deleted by injecting aav - cre into the sensorimotor cortex of floxed pten mice on postnatal day 1 .
mice were then allowed to survive for at least one year , and motor function was tested in the final months prior to euthanasia ( gutilla et al . , 2016 ) . over several months of handling and testing , mice did not exhibit any obvious behavioral abnormalities or spontaneous seizures .
general motor function was tested by open field activity and rotorod , and brains were examined extensively for any evidence of tumors or other neuropathology .
mice with pten deletion exhibited normal exploratory activity in an open field and were slightly , though not significantly , impaired on the rotorod . most important , we found no evidence of tumors or other neuropathology in the area of pten deletion .
cortical motoneurons , the cells of origin of cst axons , appeared healthy and exhibited high levels of immunostaining for the phosphorylated form of ribosomal protein s6 ( rps6 ) .
rps6 phosphorylation is considered a bioindiciator of mtor activation , so high levels of immunostaining for phosphorylated rps6 indicates continued activation of mtor more than a year after pten deletion .
the only histological abnormalities were : 1 ) cortical motoneurons lacking pten ( identified by retrograde labeling following fluorogold injections into the spinal cord ) were substantially larger than control neurons ; 2 ) there was visible disruption of the normal laminar organization of the cortex in the area of pten deletion , perhaps as a result of the increase in neuronal size ; 3 ) the ratio of neuropil to cell bodies was higher in the region of pten deletion .
our speculation is that this is due to cellular hypertrophy including hypertrophy of dendrites , but we have not yet assessed this quantitatively .
while our study does not qualify as a safety study as would be required for preclinical development of a therapy , the mice survived without any ill effects for up to 18 months after pten deletion ( considered early old age in mice ) .
other studies involving hundreds of mice and rats with pten deletion in the sensorimotor cortex report enhanced regeneration and improvements in motor function after spinal cord injury , and there have been no reports of negative effects . taken together , our findings along with previous reports point to the possibility of targeting pten therapeutically without triggering untoward effects . despite providing an important first look at the long - term consequences of pten deletion , several questions remain unaddressed .
other groups have reported seizures following deletion of pten early in development ( backman et al . , 2001 ; ogawa et al . , 2007 ;
2012 ) as well as changes in the electrophysiological properties of neurons lacking pten ( fraser et al . , 2008 ;
so far there have been no systematic studies of the physiological consequences of pten deletion in cortical neurons , but this warrants further investigation . additionally ,
since most acute neurological traumas and neurodegenerative diseases occur in adults , it will also be important to assess the consequences of pten deletion in adults .
the ability to induce a robust growth capability in central nervous system neurons has broad implications even beyond the potential of enabling regeneration of axons after spinal cord injury .
park 's original study reported that in addition to promoting axon regeneration , pten deletion in retinal ganglion cells reduced retrograde cell death following optic nerve crush .
it will be of considerable interest to assess whether pten deletion could prevent or reverse age - related deterioration of neurons such as neuronal atrophy or even prevent death that is observed in neurodegenerative diseases including parkinson 's disease , alzheimer 's disease , and amyotrophic lateral sclerosis ( als ) .
indeed , there have been reports that show that deleting pten in the substantia nigra reverses symptoms in an experimental parkinson 's disease model and protects dopaminergic neurons from toxic insults ( diaz - ruiz et al . , 2009 ; domanskyi et al . ,
the pace of research on pten related to neural regeneration is clearly accelerating . a pubmed search done on may 21 , 2016 using the keywords  pten , regeneration , and axon  in 
abstract  yielded a total of 56 papers , with an increasing number being published each year ( figure 1 ) .
this increased effort aimed at understanding pten 's role in neural regeneration will undoubtedly help to address the questions that still remain .
increased number of articles related to pten and regeneration published per year since 2006 . a pubmed search done on may 21 , 2016 using the keywords  pten , regeneration , and axon  in 
 the limited ability for injured adult axons to regenerate is a major cause for limited functional recovery after injury to the nervous system , motivating numerous efforts to uncover mechanisms capable of enhancing regeneration potential . 
 one promising strategy involves deletion or knockdown of the phosphatase and tensin ( pten ) gene . 
 conditional genetic deletion of pten before , immediately following , or several months after spinal cord injury enables neurons of the corticospinal tract ( cst ) to regenerate their axons across the lesion , which is accompanied by enhanced recovery of skilled voluntary motor functions mediated by the cst . 
 although conditional genetic deletion or knockdown of pten in neurons enables axon regeneration , pten is a well - known tumor suppressor and mutations of the pten gene disrupt brain development leading to neurological abnormalities including macrocephaly , seizures , and early mortality . 
 the long - term consequences of manipulating pten in the adult nervous system , as would be done for therapeutic intervention after injury , are only now being explored . here , 
 we summarize evidence indicating that long - term deletion of pten in mature neurons does not cause evident pathology ; indeed , cortical neurons that have lived without pten for over 1 year appear robust and healthy . 
 studies to date provide only a first look at potential negative consequences of pten deletion or knockdown , but the absence of any detectable neuropathology supports guarded optimism that interventions to enable axon regeneration after injury are achievable . 
Introduction
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