the established role of neurofilaments ( nfs ) is to increase axonal diameter in myelinated fibers thereby increasing nerve conduction velocity . nfs are composed of an n - terminal head , central rod , and c - terminal tail domain   ( figure 1 ) . nf phosphorylation , the most frequent posttranslational modification ( ptm ) and focus of our review , occurs primarily at conserved ksxxp motifs ( ksp ) located on the c - terminal tail domain of neurofilament heavy ( nf - h ) and medium ( nf - m ) [ 36 ] . however , phosphorylation of  non - ksp  serine residues within nf - m and neurofilament light ( nf - l ) amino terminal ( n - terminal ) head domain has been observed . transgenic   and gene - targeted   mouse lines expressing mutagenized nf phosphorylation sites have significantly advanced our understanding of the role of nf phosphorylation . for example , mice expressing a serine to aspartate mutation at position 55 of nf - l , nf - l , displayed accumulations of phosphorylated nfs within cell bodies . furthermore , mice expressing serine to alanine mutations within the 7 identified ksp motifs of nf - m , nf - m , demonstrated an unaltered distribution of axonal diameters .   over the last 20 years , great strides have been taken in the characterization of nf proteins , their function , and their ptms . in this review , we will attempt to describe the experiments that have led to the current understanding of the role of nf proteins and their ptms in normal and diseased axons . furthermore , using other intermediate filaments as a reference , we will detail a potential role for nf phosphorylation . moreover , we will address the importance of investigating this putative role of nf phosphorylation and its potential to explain abnormal nf accumulations in neurodegenerative diseases . axonal diameter influences the rate of neuronal conduction velocity with larger caliber axons conducting faster than small caliber axons [ 1012 ] . in myelinated axons , the internode ( myelinated ) has a larger diameter than nodes of ranvier ( unmyelinated ) . nf phosphorylation and radial growth were reduced in axons when compact myelin failed to form . moreover , phosphorylation of nfs c - termini was increased in myelinated axonal regions relative to unmyelinated regions of the same axon [ 16 , 17 ] . taken together , these data suggested that ksp repeat phosphorylation was required for establishing axonal diameter . deletion of nf - m [ 18 , 19 ] resulted in a reduction in radial growth that was similar to nerves without axonal nfs , whereas deletion of nf - h [ 2022 ] did not reduce axonal diameter to a similar level as loss of nf - l   or nf - m [ 18 , 19 ] . moreover , axonal diameter was reduced in mice expressing c - terminally truncated nf - m ( nf - m ) or c - terminally truncated nf - m and nf - h ( nf-(m / h ) )   ( figures 2(a ) and 2(b ) ) . however , truncation of nf - h ( nf - h ) only reduced axonal diameter in young mice . these data suggested that nf - m and its c - terminus were essential for radial growth and seemed to support the nf ksp phosphorylation hypothesis of radial growth . a direct test of this hypothesis was performed by expressing a ksp phosphoincompetent variant of nf - m in mice . site - directed mutagenesis was utilized to mutate all ksp serine residues to alanine preventing ksp phosphorylation ( nf - m mice ) without removing the entire c - terminus of nf - m   ( figure 2(c ) ) . surprisingly , axonal diameter was not altered in nf - m mice suggesting that ksp phosphorylation was not an essential component of radial axonal growth   ( figure 2(c ) ) . when taken together with the results obtained from nf - m mice , these results suggested that the c - terminus of nf - m mediated radial growth by a mechanism that did not require ksp phosphorylation . therefore , the precise mechanism by which nf - m c - terminus mediates radial growth has yet to be determined . although nf phosphorylation does not directly regulate radial growth , evidence suggested a progressive appearance of phosphoepitopes [ 7 , 24 , 25 ] as nfs are transported from the neuronal cell bodies to the axons . radiolabeling of nfs in retinal ganglion cells of mice suggested that nfs were more heavily phosphorylated in distal axonal regions . nf - h phosphorylation resulted in the appearance of a phosphoepitope that was recognized by the smi-34 monoclonal antibody . as nfs enter the optic nerve , this epitope was readily visualized suggesting amino acid phosphorylation that contributes to the smi-34 epitope occurred in neuronal cell bodies . a phosphoepitope recognized by smi-31 on nf - h was detected only within axons of the optic nerve subsequent to the smi-34 epitope , and the phosphoepitope recognized by rt97 antibodies was detected last and was only evident in axons of optic nerve . appearance of the rt97 epitope coincided with local accumulation of nfs and initiation of radial growth . taken together these results suggested that temporally distinct phosphorylation events of nf proteins regulated nf transport in optic nerve axons . nf accumulations are hallmarks of neurodegenerative diseases ( ndds ) . however , it is unclear how ndds alter nf dynamics resulting in the observed accumulations . analysis of nfs suggests several potential sites where disease - induced alterations could result in nf accumulations . one such mechanism is disrupted nf transport and altered local accumulation caused by altered phosphorylation dynamics . phosphorylation of nf - h and nf - m altered nf association with kinesin [ 29 , 30 ] and dynein . decreased nf - h association with the anterograde motor , kinesin , coincided with the appearance of the c - terminal rt97 phopho - epitope   and correlated with increased nf - h association with the retrograde motor , dynein . taken together , these data suggest that increased c - terminal ( ksp ) phosphorylation directed the overall flow of nf transport towards neuronal cell bodies providing evidence for a role of altered phosphorylation dynamics in the appearance of phosphorylated nf accumulations within motor neuron cell bodies . moreover , deletion of either nf - h [ 1921 ] or nf - m   increased the rate of nf transport in vivo supporting a role for ksp phosphorylation in regulating nf transport . therefore , increased rates of nf transport may have resulted from increased association of nfs with kinesin , if loss of a single subunit results in a net loss of phosphorylation . interestingly , deletion of nf c - termini and all ksp repeats did not alter nf transport rates [ 22 , 33 , 34 ] . unaltered rates of nf transport in mice expressing truncated nfs appeared to contradict a primary role for nf phosphorylation in regulating nf transport . however , truncation of a single subunit resulted in compensatory phosphorylation of the remaining subunit [ 22 , 23 ] . thus , phosphodependent regulation of nf transport can not be completely ruled out until transport is measured in mice simultaneously expressing c - terminally truncated nf - m and nf - h . nf - l   and nf - m   were phosphorylated at serine residues located throughout their amino termini , especially serine . mimicking constitutive phosphorylation by mutating serine residues to aspartate , such as nf - l , altered nf assembly and decreased nf transport . transgenic mice expressing low levels of nf - l developed nf accumulations within neuronal cell bodies in mice as young as 4 weeks old . additionally , phosphorylation of nf - m at protein kinase a sites within the n - terminus inhibited c - terminal ksp phosphorylation of nf - m . thus , phosphorylation of nf - l and nf - m on serine residues located within the n - terminus may be a mechanism to delay nf assembly and phosphorylation thereby preventing ectopic accumulation of nfs within neuronal cell bodies . altering nf subunit stoichiometry by overexpression of nf - l [ 36 , 37 ] , nf - l and nf - h overexpression resulted in phosphorylated nf accumulations in motor and sensory neuron cell bodies that were not observed in control littermates [ 37 , 38 ] . nf - m overexpression resulted in decreased nf - h phosphorylation and increased nf - l expression . furthermore , nf - l or nf - h overexpression resulted in morphological abnormalities , cell body swelling , and muscle atrophy that were similar to the pathologies observed in amyotrophic lateral sclerosis ( als ) [ 39 , 40 ] . neuronal pathology was alleviated in nf - h transgenic mice by simultaneous overexpression of nf - l   highlighting the importance of regulating the relative stoichiometry of the individual nf subunit proteins . nf - l mrna expression was also reduced in sporadic   and superoxide dismutase 1-linked familial als . deletion of nf - l in mice resulted in accumulation of nf - h and nf - m in neuronal cell bodies . if reductions in nf - l mrna expression lead to reduced nf - l protein , then , as observed in genetically altered mice , altering nf subunit stoichiometry may contribute to nf accumulations observed in both ad and als . a potential mechanism for regulating nf content within the axon is degradation by the ubiquitin proteasome system ( ups ) [ 4648 ] . disruption of two components of the ups , tripartite motif protein 2 ( trim2 ) mice   and ubiquitin - specific protease 14 ( usp14 ) ( ax mice ) [ 4951 ] , resulted in phenotypic ataxia resulting from motor neuron dysfunction . upon further examination , trim2 mice displayed alterations associated with neurodegeneration . one - month - old trim2 mice contained axonal swellings and nf accumulations in regions that overlapped calbindin d-28k immunoreactivity suggesting that the axons originated from purkinje neurons . by 5 months , trim2 mice showed an 85% reduction in the number of purkinje cells relative to control mice . ax mice demonstrated a selective loss of large caliber axons within the fourth and fifth lumbar ( l4-l5 ) motor roots . furthermore , ax mice displayed severe neuromuscular junction ( nmj ) defects such as swollen and poorly arborized nerve terminals that demonstrated sprouting , denervation , and disorganized postsynaptic achrs . within the tibialis anterior ( ta ) muscle , axonal swellings contained nf accumulations that were observed as early as p7 . ax mice had a 1.5-fold increase in miniature end - plate potential ( mepp ) amplitude . additionally , mepp frequency and vesicular quantal content were both reduced by ~40% suggesting synaptic vesicle disorganization [ 49 , 50 ] . disruptions to both components of the ups resulted in axonal swellings containing phosphorylated nf accumulations . moreover , restoration of neuronal usp14 levels ( thy1-usp transgene ) in ax mice alleviated phenotypic ataxia   and nf accumulations . therefore , altering the neuronal ups resulted in nf accumulations that were similar to those observed in mice expressing nfs that mimic constitutive phosphorylation within the n - terminal head domain , mice with altered nf stoichiometry , and many human ndds . nfs are long - lived axonal proteins whose accumulation within the axon is strictly regulated . although evidence suggests that nfs associate with molecular motors [ 26 , 29 , 30 ] in a phosphodependent manner to reach their destination , little is known about the mechanisms that prevent excessive nf accumulation and regulate local nf turnover . the appearance of nf accumulations in axons with ups defects suggests that the ups has a critical role in the regulation of nf turnover . ubiquitin epitopes have been identified on nf - m purified from spinal cord verifying that ubiquitination is indeed nf ptm . furthermore , nf - l has been identified as a target of the e3 enzyme , trim2 , and nf - m has been identified as a target of the e3 enzyme , carboxyl - terminus of hsc70 interacting protein ( chip ) . identification of nf e3 enzymes supports the claim that nfs may undergo ubiquitin - mediated degradation . however , the mechanisms responsible for regulating nf ubiquitination have yet to be determined . trim2 and chip are classified as e3 ligases [ 46 , 47 ] which are proteins that catalyze the transfer of ubiquitin to substrate proteins . both trim2   and chip   targeted and ubiquitinated protein substrates in a phosphodependent manner . the e3 ligase , c - cbl , had a similar phosphodependence for its interaction with epidermal growth factor receptor ( egfr ) and sprouty-2 ( spry2 ) . although evidence suggested that trim2 and chip require substrate phosphorylation [ 54 , 55 ] , it is unknown if increased phosphorylation would negatively regulate trim2 and chip affinity for nf subunits similar to c - cbl . nf phosphorylation was increased in cells   and brain slices   treated with the phosphatase inhibitor , okadaic acid , resulting in increased nf levels [ 57 , 58 ] . furthermore , treating purified nfs with alkaline phosphatase resulted in dephosphorylated nfs that were degraded more rapidly by the calcium dependent protease , calpain , when compared to phosphorylated nfs . therefore , increased nf phosphorylation may be a mechanism for enhancing nf stability by preventing nf turnover by ups or protease - dependent mechanisms . ubiquitination of the type i / ii intermediate filaments , keratins , may offer insight into the interaction between nf phosphorylation and ubiquitination . like nfs , a proline to leucine mutation at position 74 of keratin 8 , k8 , eliminated the phosphorylation of this consensus site and increased k8 ubiquitination . these data suggest that phosphorylation and ubiquitination of k8 are reciprocally regulated [ 61 , 62 ] . the similarity between nfs ( type iv intermediate filaments ) and keratins ( type i / ii intermediate filaments ) suggests that nf ksp phosphorylation could regulate subsequent ptms in a manner similar to keratin phosphomotifs . in many ndds , accumulations of phosphorylated nfs are hallmarks of pathology . increased phosphorylation resulted in increased nfs [ 57 , 58 ] and decreased k8 ubiquitination , whereas dephosphorylation of the most heavily phosphorylated nf subunit , nf - h , increased its turnover sixfold . furthermore , nf - l   and nf - m   have recently been identified as e3 ligase targets . taken together , these data suggest that ectopic phosphorylation of nfs results in nf accumulations , which may lead to further phosphorylation subsequent to aggregate formation . delineating the mechanisms that regulate nf phosphorylation may prove to be critical in regulating nf accumulations in ndds . in this section als is an adult - onset ndd that selectively kills upper and lower motor neurons . despite common pathogenic features , there is no identifiable genetic linkage in 9095% of als cases ( sporadic als ) while 510% of als occurrences result from dominantly inherited genetic mutations ( familial als ) . dominant missense mutations in the gene for the cytoplasmic cu / zn superoxide dismutase 1 ( sod1 ) are responsible for 20% of familial als cases . motor neuron cell bodies contain nf accumulations in cases of sporadic   and familial als . mice expressing sod1-linked mutations resulted in motor neuron pathology that included ectopic accumulations of phosphorylated nfs within lumbar motor neuron cell bodies . furthermore , sod1-linked als mouse models demonstrated impaired slow axonal transport of nfs as the earliest indication of pathology . in order to identify the role of nfs in sod1-linked als pathogenesis the absence of assembled nfs in axons ( nf - l ) alleviated the selective motor neuron vulnerability and slowed sod1-mediated toxicity . furthermore , elimination of nf ksp motifs via single or double truncation of nf - m and nf - h c - termini delayed disease onset and extended survival of the sod1 transgenic mouse model . interestingly , delayed onset and enhanced survival were additive with truncation of both nf - m and nf - h c - termini enhancing survival better than truncation of a single nf c - terminus . furthermore , preventing nf ksp phosphorylation through c - terminal truncation of nf - m and nf - h enhanced motor neuron survival in sod1 mice . these results suggested that the nf network , particularly nf - m and nf - h c - termini and their phosphorylation , contributes to als pathogenesis . however , the mechanism by which removal of axonal nfs or preventing their c - terminal phosphorylation ameliorated als pathology remains to be determined . based upon the specific genetic defect cmts are grouped into four main types , cmt1 - 4 with each type having several subtypes [ 68 , 69 ] . cmt2e has been linked to mutations throughout the functional domains of nf - l   ( figure 3(a ) ) . nf - l mutations are typically autosomal dominant though two recessive forms have been identified [ 71 , 72 ] ( figure 3(a ) ) . cmt2e two cmt2e - linked nf - l mutations are located at positions , pro and pro . these mutations are located adjacent to known bovine head domain phosphorylation sites , ser , ser , ser , and ser(figure 3(b ) ) . interestingly , pro and pro along with all n - terminal serine residues are highly conserved across mammals   ( figure 3(b ) ) . all but one of these sites ( ser ) have complete sequence homology in nf - l from a variety of mammals   ( figure 3(b ) ) . when expressed in cultured cells , the hnf - l mutation abolished nf - l head domain phosphorylation . additionally , mutation of a proline residue in keratin 8 prevented phosphorylation of a nearby serine residue . taken together , these data suggest that nf - l and nf - l mutations may prevent n - terminal phosphorylation . as previously discussed , expressing nf - l that mimics constitutive phosphorylation of an n - terminal serine , nf - l , prevented nf assembly , and n - terminal phosphorylation of nf - m prevented c - terminal phosphorylation . based on the previously described role of nf phosphorylation , abolished n - terminal phosphorylation would result in ectopic nf assembly and phosphorylation [ 7 , 8 , 75 ] , altered nf transport [ 21 , 26 , 30 , 33 , 35 ] , and ectopic nf accumulations [ 7 , 8 , 75 ] . consistent with this prediction , transgenic mice expressing the cmt - linked head domain mutation , hnf - l , displayed disrupted axonal transport . furthermore , mutations within a conserved sequence at the end of the rod domain , hnf - l , also resulted in ectopic nf phosphorylation as early as 1 month . sma is an autosomal recessive disorder that is the leading genetic cause of infantile death . sma is caused by a deficiency in survival motor neuron ( smn ) protein levels produced by the smn1 gene [ 79 , 80 ] , which is a ubiquitously expressed protein that has a well - described role in rna metabolism [ 8185 ] . the severity of sma is dependent on the relative copy number of the smn2 gene which produces ~15% functional smn that compensates for the loss of smn1 [ 87 , 88 ] . as a result , sma has a broad disease spectrum made up of sma type 0 , i , ii , iii , and iv . within motor neurons , similar nf accumulations were one of the earliest pathological alterations observed in sma mouse models [ 9194 ] . however , recent work demonstrated that nf accumulations were likely a result of local nf alterations . ectopic nf accumulations within motor neuron cell bodies were not apparent early or late in disease . moreover , the nf network was not altered in proximal segments of motor axons of the fifth lumbar ventral root , which is one of three lumbar segments that together form the sciatic nerve . interestingly , mice with alterations to the ups displayed phosphorylated nf accumulations   strikingly similar to those observed in sma models [ 9093 ] . taken together , these results suggest that mechanisms disrupting nf turnover within motor neurons may be responsible for nf accumulations within nmj of sma patients . one potential nf alteration that could lead to reduced nf turnover and subsequent accumulation is altered nf phosphorylation . phosphorylation of nfs was initially documented nearly 30 years ago and thought to regulate radial growth within myelinated axons . however , a series of recent analyses conducted on genetically modified mice has provided evidence against the role of nf phosphorylation in radial growth . current evidence suggests that nf phosphorylation is both spatially and temporally regulated , which may be a mechanism to regulate nf assembly and accumulation . the presence of phosphorylated nfs in human ndds suggests that altered nf phosphorylation dynamics may contribute to aberrant nf accumulation . therefore , understanding the role of and mechanisms regulating nf ptm may prove critical to our understanding of the development and functioning of the nervous system in both healthy and diseased neurons .
posttranslational modification of proteins is a ubiquitous cellular mechanism for regulating protein function . 
 some of the most heavily modified neuronal proteins are cytoskeletal proteins of long myelinated axons referred to as neurofilaments ( nfs ) . 
 nfs are type iv intermediate filaments ( ifs ) that can be composed of four subunits , neurofilament heavy ( nf - h ) , neurofilament medium ( nf - m ) , neurofilament light ( nf - l ) , and -internexin . within wild type axons , nfs are responsible for mediating radial growth , a process that determines axonal diameter . 
 nfs are phosphorylated on highly conserved lysine - serine - proline ( ksp ) repeats located along the c - termini of both nf - m and nf - h within myelinated axonal regions . 
 phosphorylation is thought to regulate aspects of nf transport and function . 
 however , a key pathological hallmark of several neurodegenerative diseases is ectopic accumulation and phosphorylation of nfs . 
 the goal of this review is to provide an overview of the posttranslational modifications that occur in both normal and diseased axons . 
 we review evidence that challenges the role of ksp phosphorylation as essential for radial growth and suggests an alternative role for nf phosphorylation in myelinated axons . 
 furthermore , we demonstrate that regulation of nf phosphorylation dynamics may be essential to avoiding nf accumulations .