Specialized Roles of Neurofilament Proteins in Synapses

Home , Internexin, Spectrin

Brain Research Bulletin 126 (2016) 334–346

Contents lists available at ScienceDirect

Brain Research Bulletin

j ournal homepage: www.elsevier.com/locate/brainresbull

Review

Specialized roles of neuroﬁlament proteins in synapses: Relevance to

neuropsychiatric disorders

a,b,∗ a,b,c,∗

Aidong Yuan , Ralph A. Nixon

Center for Dementia Research, Nathan Kline Institute, Orangeburg, New York, 10962, United States

Departments of Psychiatry, New York University School of Medicine, New York, NY, 10016, United States

Department of Cell Biology, New York University School of Medicine, New York, NY, 10016, United States

a r t i c l e i n f o a b s t r a c t

Article history: Neuroﬁlaments are uniquely complex among classes of intermediate ﬁlaments in being composed of four

Received 11 July 2016

subunits (NFL, NFM, NFH and alpha-internexin in the CNS) that differ in structure, regulation, and func-

Received in revised form 2 September 2016

tion. Although neuroﬁlaments have been traditionally viewed as axonal structural components, recent

Accepted 3 September 2016

evidence has revealed that distinctive assemblies of neuroﬁlament subunits are integral components

Available online 5 September 2016

of synapses, especially at postsynaptic sites. Within the synaptic compartment, the individual subunits

differentially modulate neurotransmission and behavior through interactions with speciﬁc neurotrans-

Keywords:

mitter receptors. These newly uncovered functions suggest that alterations of neuroﬁlament proteins

Neuroﬁlament subunit

Synapse not only underlie axonopathy in various neurological disorders but also may play vital roles in cog-

nition and neuropsychiatric diseases. Here, we review evidence that synaptic neuroﬁlament proteins

Dendritic spine

Neuropsychiatric disease are a sizable population in the CNS and we advance the concept that changes in the levels or post-

translational modiﬁcation of individual NF subunits contribute to synaptic and behavioral dysfunction

in certain neuropsychiatric conditions.

Contents

1. Introduction ...... 334

2. NF proteins in synapses ...... 335

2.1. Distinctive assemblies of NF subunits in synapses ...... 335

2.2. NF subunit-speciﬁc modulation of synaptic functions and behavior ...... 338

3. Alterations of NF proteins in psychiatric disorders ...... 339

3.1. Schizophrenia and bipolar disorder ...... 339

3.2. Drug addiction ...... 340

4. Alterations of NF proteins in Alzheimer’s disease ...... 341

5. Conclusion ...... 342

Acknowledgements ...... 342

References ...... 342

1. Introduction

homopolymers, NFs in the CNS are hetero-polymers composed of

Neuroﬁlaments (NFs), the intermediate ﬁlaments of mature

NFL, NFM, NFH and alpha-internexin subunits (Yuan et al., 2006).

neurons, are among the most abundant proteins in brain. Unlike

Although structurally distinctive, these four NF subunits share a

the intermediate ﬁlaments of other cell types, which are usually

basic tripartite domain structure consisting of a conserved cen-

tral ␣-helical rod region, a short variable head domain at the

∗ amino-terminal end and a tail of highly variable length at the C-

Corresponding authors at: Center for Dementia Research, Nathan Kline Institute,

terminal end. The short head domain is rich in serine and threonine

Orangeburg, New York, 10962, United States

residues and contains consensus sites for O-linked glycosylation

E-mail addresses: [email protected] (A. Yuan), [email protected]

(R.A. Nixon). and phosphorylation (Yuan et al., 2012a). The central rod domain,

http://dx.doi.org/10.1016/j.brainresbull.2016.09.002

A. Yuan, R.A. Nixon / Brain Research Bulletin 126 (2016) 334–346 335

which is relatively conserved among intermediate ﬁlament family the cytoskeleton in synapses is composed of microtubules, actin

members, contains long stretches of hydrophobic heptad repeats ﬁlaments, the spectrin-rich membrane skeleton, and as recently

favoring formation of ␣-helical coiled-coil dimers. The C-terminal shown, NF assemblies (Yuan et al., 2015b, 2015c). Evidence is also

domains contain glutamic- and lysine-rich stretches of varying emerging that the cytoskeleton, and especially the NF scaffold, acts

length that mainly establish the size range (58–200 kDa on SDS as a docking platform to organize the topography of organelles

gels) of the four NF subunits. Although all intermediate ﬁlament within different neuronal compartments. Rearrangements of this

types serve roles as structural scaffolds, NF subunit heterogeneity topography are dynamically coordinated at least in part by cellular

also confers specialized structural properties to NF, in axons where signals regulating the phosphorylation state of the binding part-

the ﬁlaments are extremely long and are often arranged in paral- ners (Perrot and Julien, 2009; Rao et al., 2011; Styers et al., 2004;

lel with uniform spacing conferred by the long C-terminal tails of Yuan et al., 2015b). Such evidence suggests a range of possibili-

NFM and NFH extending perpendicularly from the ﬁlament core ties for understanding the newly recognized roles of individual NF

(Rao et al., 2003, 2002). These unique space ﬁlling properties of NF subunits in modulating synaptic function. In this review, we con-

facilitate their well-established role in caliber expansion of large- sider the properties of synaptic NF assemblies in the CNS and, in

diameter myelinated axons of peripheral nerves, which is critical this context, raise the possibility that certain changes in levels or

for effective nerve conduction (Zhu et al., 1997). NFs also exten- phosphorylation of NF subunits reported in neuropsychiatric dis-

sively cross-link with other cytoskeletal elements along axons to orders (Table 1) disrupt synaptic signaling or, in some cases, reﬂect

form a large metabolically stable stationary NF network (Nixon and adaptive or maladaptive responses to these synaptic disruptions.

Logvinenko, 1986; Yuan et al., 2015a, 2009) that is critical to axon

caliber expansion (Friede and Samorajski, 1970; Hoffman et al.,

1987; Ohara et al., 1993) and organelle distribution along axons 2. NF proteins in synapses

(Rao et al., 2011).

Besides these unique structural and functional features, NF sub- 2.1. Distinctive assemblies of NF subunits in synapses

units are distinguished from other intermediate ﬁlament proteins

by the complex regulation of their head and tail domains by phos- Synapses have long been considered to be degradative sites for

phorylation, especially those of NFM and NFH, which involves NF reaching terminals by axonal transport (Roots, 1983). When

actions of multiple protein kinases and phosphatases at many NF proteins have been detected in synaptic fractions and bound

polypeptide sites (Pant and Veeranna, 1995). Although certain to synaptic proteins in vitro, they have previously been viewed

phosphorylation events are known to control tail extension and as contaminating axonal NF proteins (Matus et al., 1980) and

subunit assembly and slow turnover, the purpose of such complex their possible role in synapses has rarely been entertained. Using

and dynamically changing phosphate topography on NF subunits multiple independent approaches, however, Yuan et al. recently

(de Waegh et al., 1992; Nixon and Sihag, 1991; Pant and Veeranna, provided deﬁnitive evidence that all four CNS NF subunits are inte-

1995) has remained puzzling, given the mainly static structural gral resident proteins of synapses and have distinct roles in synaptic

support roles ascribed to NF. Both the complex hetero-polymeric function and behavior (Fig. 1)(Yuan et al., 2015b, 2015c). The NF

structure and dynamically changing phosphate topography of NF assemblies isolated from synapses are distinctive as compared to

proteins suggests that individual NF proteins might serve addi- those in other parts of the neuron in terms of their morphology

tional biological roles although there has been relatively little and in having higher proportions of alpha-internexin, a lowered

exploration of this issue until recently. phosphorylation state of NFM, and a higher NFH phosphorylations

NF gene mutations are well recognized as causes of several state. This unique population of synaptic NF proteins is more abun-

neurological disorders mainly involving degeneration of periph- dant in the postsynaptic area than in adjacent dendritic areas or

eral nerve ﬁbers in accordance with the prominent function of NF presynaptic terminals and exhibits a different response to subunit

in supporting large-diameter myelinated axons (Brownlees et al., perturbation than the axonal population. For example, when NFM is

2002). Notably, however, NF proteins are abundant in grey matter deleted from mice, NFH phosphorylation and the ratio of NFH to NFL

CNS regions as well as white matter (Chan et al., 1997) but inﬂu- subunit increase signiﬁcantly in the synaptic pool in comparison to

ence caliber expansion much less dramatically in most populations the total NF pool (Yuan et al., 2015b). Because NF subunit monomers

of CNS axons (Dyakin et al., 2010). These observations and evidence are inefﬁciently transported along axons (Yuan et al., 2003, 2006,

that NF subunits can be axonally transported in various minimally 2015b), NF proteins in synapses are at least oligomeric in form and

assembled forms (including as heterodimers) suggest a broader dis- can be transported as such in axons, providing one possible basis

tribution and range of assembly forms of NF subunits within CNS for their synaptic location. It is also possible that some NF protein

neurons, including substantial populations in synapses. In light of in postsynaptic boutons is synthesized locally because mRNAs of

these ﬁndings, alterations of a particular NF subunit as seen in NFL (Crino and Eberwine, 1996; Paradies and Steward, 1997) and

speciﬁc brain regions in psychiatric and neuropsychiatric disor- alpha-internexin (Villace et al., 2004) are reported to be present in

ders (Cairns et al., 2003; Clinton et al., 2004; Garcia-Sevilla et al., dendrites and polyribosomes are preferentially localized under the

1997) may reﬂect an alteration within synapses which inﬂuences base of dendritic spines (Steward and Levy, 1982). In this regard,

the clinical phenotype. evidence suggests that NF proteins identiﬁed in nerve terminals

Psychiatric diseases, affecting an estimated 54 million Amer- isolated from squid brain are produced by local protein synthesis

icans yearly, cause mild to severe disturbances in thought or (Crispino et al., 1993; Martin et al., 1990). In light of these ﬁndings,

behavior usually in the absence of known changes in axonal aforementioned reports noting the presence of NF in synaptic frac-

integrity. Nevertheless, changes in levels and phosphorylation of tions and interactions of NF proteins with known synaptic proteins

NF subunits have consistently been noted in certain psychiatric can be viewed as further support for a substantial synaptic pool of

disorders although the location of these changes within neurons is NF proteins in the brain.

poorly understood. Psychiatric diseases prominently involve alter- Remarkably, previous proteomic analyses of synaptic fractions

ations of synaptic transmission. The highly specialized composition have consistently identiﬁed NF proteins but these ﬁndings were not

of synapses includes not only the well characterized vesicular and followed up with additional proof of a synaptic localization or func-

protein receptor machinery supporting neurotransmission but also tion. During the study of activity-regulated proteins in postsynaptic

a specialized cytoskeleton important for delivering, inserting, and density fractions by two-dimensional gel electrophoresis and mass

recycling synaptic components. Like other domains in a neuron, spectrometry, Satoh et al. identiﬁed a total of 90 spots contain-

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IHC IHC

peptide WB Proteomic Proteomic Proteomic Proteomic Proteomic Proteomic Prpteomic Proteomic Proteomic Proteomic Proteomic WB IHC WB IHC WB IHC IHC WB IHC IHC WB IHC WB WB, WB IHC WB WB, IHC Proteomic

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cortex

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Brain DLPFC ACC CC DLPFC DLPFC CC DLPFC ACC CC DLPFC DLPFC DLPFC DLPFC DLPFC DLPFC VTA VTA Frontal Hippocampus VTA VTA Hippocampus VTA VTA Hippocampus Occipital Sciatic Hippocampus Hippocampus Frontal Sciatic Hippocampus Hippocampus Frontal Temporal Cortex

immunohistochemistry;

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Increase Increase Decrease Decrease Increase Increase Increase Decrease Increase Increase Increase Increase blotting;

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Regulation Protein Decrease Decrease Decrease Decrease Decrease Decrease Decrease Increase Increase Decrease Decrease Increase Increase Decrease Decrease Decrease Decrease Decrease Decrease Decrease Decrease Decrease Decrease Decrease Decrease Decrease Decrease Increase callosum;

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Expression

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addiction

1 DLPFC,

: Disease Schizophrenia Drug Bipolar Alzheimer Table Note Neuroﬁlament

A. Yuan, R.A. Nixon / Brain Research Bulletin 126 (2016) 334–346 337

Fig. 1. Functional NF subunit assemblies in synapses. Left panel: Immunogold labeled antibodies against the NFM subunit decorating synaptic structures in a linear pattern

(immunogold particles outlined in blue) suggesting the presence of short NFs and protofilament/protofibril or unit length filament assemblies. In the upper inset, a filament

within a postsynaptic bouton is decorated by immunogold antibodies to both NFL (large gold dots) and NFH (small gold dots). Morphometric analysis indicates a higher

density of immunogold labeling in postsynaptic boutons than in preterminal dendrites or presynaptic terminals (graph inset). Middle panel: Ultrastructural image of a

human synapse depicts membranous vesicles, many of which appear to be associated with a loose network of short 10 nm ﬁlaments in the post-synaptic region. Right panel:

Evidence supports a biological mechanism whereby D1 dopamine receptors internalized on endosomes from the postsynaptic surface (red asterisks) dock on synaptic NF

subunit assemblies (outlined in blue) where they are readily available to recycle on endosomes to the surface in response to ligand stimulation (adapted from Yuan et al. Mol

Psychiatry 2015; 20:915 with permission).

ing 47 different protein species in the PSD fractions isolated from (Benson et al., 1996). This immunoreactivity receded from spines

mouse forebrain (Satoh et al., 2002). Among these spots, alpha- and remained at the base of dendritic protrusions when cells were

internexin and NFM accounted for 8 and 4 spots, respectively. With treated with latrunculin A to disrupt actin ﬁlaments (Zhang and

an improved proteomic method involving nanoﬂow HPL coupled Benson, 2001).

to electrospray tandem mass spectrometry (LC–MS/MS), Jordan Besides their association with the PSD, NF subunits have been

et al. identiﬁed all four CNS NF proteins (NFL, NFM, NFH and alpha- noted to interact with other synaptic proteins. The synapse-speciﬁc

internexin) as abundant components of biochemically puriﬁed PSD PSD95-associated protein SAPAP interacts via its N-terminal region

fractions from rat or mouse brain(Jordan et al., 2004). A subsequent with all 4 NF subunits, co-immunoprecipitates with NF proteins

phospho-proteomic analysis of PSD proteins, identiﬁed 42 phos- from rat brain, and co-localizes with NFs in transfected COS cells

phoproteins in PSD preparations from mouse brains (Trinidad et al., (Hirao et al., 2000). Spinophilin, a synaptic protein highly enriched

2005). Out of 90 phosphorylated peptides derived from these 42 in dendritic spines (Allen et al., 1997), regulates the formation and

phosphoproteins, NF proteins accounted for 23 (NFL 1, NFM 5 and function of the spines (Feng et al., 2000). Using a shotgun pro-

NFH 17), indicating the considerable abundance of NF proteins in teomic approach, Baucum et al. showed that spinophilin interacts

the PSD. NF proteins are also glycosylated and in a glyco-proteomic via its N-terminal domain with NFL, NFM, and alpha-internexin in

analysis of PSD preparations from mouse brain, NFL, NFM and mouse striatum (Baucum et al., 2010, 2012; Hiday, 2015). More-

alpha-internexin were among 18 identified GlcNAc-modified pro- over, the association increased significantly during mouse brain

teins (Vosseller et al., 2006). A limited idea of how NF proteins are maturation (Baucum et al., 2012) during the period of synapto-

organized within the PSD has been achieved using a novel solid genesis (Aghajanian and Bloom, 1967) and was dependent on the

phase and chemical crosslinking approach to distinguish proteins phosphorylation state of either spinophilin or NF protein (Hiday,

that reside either at the surface of the PSD or in its interior (Liu et al., 2015). The NFL subunit also directly binds to tau (Miyata et al.,

2006). The analysis suggested that NFM, NFH and alpha-internexin 1986), a protein initially considered to localize in dendritic spines

together with tubulin subunits reside in the interior of the PSD only under pathological conditions (Hoover et al., 2010), but is

while actin resides primarily on the surface. Recently, Moczulska not believed to have a physiological role in dendritic functioning

et al. performed precise quantiﬁcation of the mouse synaptoso- (Frandemiche et al., 2014; Ittner et al., 2010). Knockdown of tau

mal proteome during postnatal maturation (from 3 − 8 weeks of using speciﬁc shRNA signiﬁcantly decreased the spine density of

age) using peptide-based iTRAQ labeling and high-resolution two- cultured hippocampal neurons (Chen et al., 2012) and deletion of

dimensional peptide fractionation (Moczulska et al., 2014). Among tau in mice abolished long-term depression (Kimura et al., 2014).

the 3422 identiﬁed proteins with complete quantiﬁcations, NFL, Thus the interaction between NFL and tau in dendritic spine could

NFM, NFH and alpha-internexin were established to be components be important for normal synaptic transmission.

of the synaptosomes and their levels increased during brain postna- NF polymers are highly abundant in myelinated axons as esti-

tal maturation, a period when synapse formation sharply increases mated by conventional electron microscopy but 10 nm ﬁlaments

(Aghajanian and Bloom, 1967). Alpha-internexin had long been have also been noted in the perikarya and dendrites of neurons,

believed to form a separate ﬁlament system from NF triplet proteins including ones lacking a myelinated axon (Dyakin et al., 2010;

until it was shown to be the fourth subunit of NFs in the mature CNS Hirokawa et al., 1984; Kong et al., 1998; Zhang et al., 2002). In

(Yuan et al., 2006) although somewhat over-represented among the 1977, Blomberg et al. reported 10 nm ﬁlaments as major con-

other NF subunits present in synapses (Yuan et al., 2015b). Ben- stituents of the partially broken up PSD isolated from dog cerebral

son et al. detected alpha-internexin immunoreactivity in dendrites cortex and assumed that they were composed of NF proteins

extending into dendritic spines of cultured hippocampal neurons (Blomberg et al., 1977). Using a rapid-freezing technique, Landis

338 A. Yuan, R.A. Nixon / Brain Research Bulletin 126 (2016) 334–346

and Reese described infrequent 9–10 nm ﬁlaments in dendritic et al. recently determined LTP in the Schaffer collateral path-

spines of mouse brain but interpreted these structures as actin ﬁla- way of the hippocampal slices prepared from wild-type control

ments due to their periodicity (Landis and Reese, 1983). Yuan et al. and IHL-TKO mice lacking alpha-internexin, NFH and NFL sub-

also identiﬁed in some synapses that short 10 nm ﬁlaments were units (Yuan et al., 2015b). The tetanic stimulation evoked a typical

decorated by immunogold antibodies to different NF subunits indi- LTP of fEPSP in slices from wild-type mice. These responses were

cating that at least some synaptic NF subunits exist in polymerized stable for 120 min. However, tetanic stimulation evoked a signiﬁ-

form (Yuan et al., 2015b, 2015c). Although NF proteins are present cantly reduced fEPSP slopes in IHL-TKO mice, indicating NF proteins

in both pre- and postsynaptic areas, 10 nm NF polymers are not are, therefore, required for synaptic plasticity. Because LTP in the

abundant in synaptic areas compared to myelinated axons, esti- Schaffer collateral pathway of the hippocampus is NMDA receptor-

mated by conventional electron microscopy. The infrequency of dependent, altered LTP may be associated with reduced levels

conventional long intermediate-sized polymers in synapses indi- of NMDA-NR1 receptor (Yuan, Veeranna et al. unpublished data).

cates that most NF subunits in synapses are probably in the form Yuan et al. also conducted a 5-trial social memory assay to deter-

of oligomeric structures (protoﬁlaments or protoﬁbrils). Consistent mine if IHL-TKO mice have a social memory defect. In this test,

with this interpretation, transport of NFM in non-ﬁlamentous, but the subject mouse was given four brief exposures (trials 1–4) in its

oligomeric form, has been reported (Terada et al., 1996; Yabe et al., home cage to the same stimulus mouse (intruder). In the 5th trial,

1999; Yuan et al., 2003). The organization of oligomeric NF assem- the subject mouse encountered an entirely novel stimulus mouse

blies in synapses, quite different from those parallel arrays with (novel intruder). Wild-type control mice displayed normal social

spacing controlled by C-terminal extensions of NFH/MFM in axons, memory, as demonstrated by a marked habituation (decreased

might be closely associated with the synaptic actin network since exploration) during the ﬁrst 4 trials and a striking dishabituation

disruption of such actin ﬁlaments also remarkably affects the dis- (increased exploration) on the presentation of a novel animal on the

tribution of NF assemblies (Zhang and Benson, 2001). The relative 5th trial. In contrast, IHL-TKO mice showed no signiﬁcant habitua-

abundance of NF proteins in synapses is synapse type-dependent, tion during the 4 exposures to the stimulus mouse or dishabituation

e.g., NF proteins are not present at the postsynaptic sites of neuro- to the novel stimulus mouse. Consistent with reduced LTP, IHL-TKO

muscular junctions. Instead, another intermediate ﬁlament protein showed social interaction deﬁcits, indicating that mice lacking NF

desmin is highly concentrated near the AChR-rich crests of the junc- proteins failed to develop normal social memory.

tional folds at the neuromuscular junctions (Sealock et al., 1989). Dopamine receptors are G-protein-coupled receptors that

mediate functions of dopamine ranging from voluntary move-

2.2. NF subunit-speciﬁc modulation of synaptic functions and ment and reward to hormonal regulation and hypertension (Girault

behavior and Greengard, 2004). These receptors are highly concentrated in

the postsynaptic membrane of dopaminergic synapses. Yeast-two-

N-methyl-d-aspartate (NMDA) receptors are excitatory neuro- hybrid screening has shown that the C-terminal tail of NFM directly

transmitter receptors critical for synaptic plasticity and neuronal interacts with the cytoplasmic loop of dopamine D1receptor (Kim

development in the mammalian brains (Li and Tsien, 2009). These et al., 2002) and that their co-expression in HEK-293 cells resulted

receptors are highly concentrated in postsynaptic membranes of in more than 50% reduction of receptor binding. Recently, the rele-

glutamatergic synapses. Yeast-two-hybrid screening revealed that vance of this interaction at synapses was established in genetically

NFL interacts through its rod domain directly with the cytoplasmic modiﬁed mice. Deletion of NFM, but not deletion of any of the other

C-terminal domain of NR1 (Ehlers et al., 1998) suggesting that NFs NF subunits, greatly ampliﬁed dopamine D1-receptor-mediated

may anchor or localize NMDA receptors within the neuronal plasma motor responses to cocaine while redistributing postsynaptic D1-

membrane. Co-expression of NFL with either NR1 or NR2B subunits receptors from endosomes to the plasma membrane (Fig. 2)(Yuan

in HEK293 cells did not increase surface expression of these sub- et al., 2015b). In wild-type mice, NFM colocalized with the D1

units whereas expression of all three components increased surface receptor in synaptic boutons by immunogold electron microscopy.

abundance of NR1 by 20% and concomitantly increased NMDA- Deletion of NFM also signiﬁcantly increased D1R-stimulated hip-

mediated cytotoxicity (Ratnam and Teichberg, 2005). The NR1 pocampal LTP further indicating an in vivo interaction between

subunit is ubiquitinated in HEK293 cells and the co-expression with NFM and D1R (Yuan et al., 2015b). Depressed hippocampal LTP

NFL antagonizes this ubiquitination process, suggesting another induction is NF subunit-selective: maintenance of LTP is deﬁcient

way that the interaction of NFL with NR1 may stabilize the NMDA in NFH-null mice while basal neurotransmission and induction of

receptor. NFL also binds to protein phosphatase-I (PP1), a pro- LTP are normal in NFM-null mice. Eliminating all NF proteins from

tein/serine/threonine phosphatase in the PSD (Terry-Lorenzo et al., the CNS profoundly disrupted synaptic plasticity and social mem-

2000). NFL-bound PP1 could regulate the phosphorylation states ory without altering the structural integrity of synapses (Yuan et al.,

of NF subunits and NMDA NR1 receptors the cellular distribution 2015b).

of which is regulated by the phosphorylation of speciﬁc serines Peripherin, a subunit of peripheral nerve NFs, is almost exclu-

in the C1 exon cassette (Ehlers et al., 1995). On the one hand, sively expressed in the neurons of PNS and in the CNS only in

NFL may modulate synaptic plasticity through interactions with deﬁned subsets of neurons directly projecting to the periphery

NR1, PP1 or 14-3-3 (Ehlers et al., 1998; Miao et al., 2013; Terry- (Brody et al., 1989; Yuan et al., 2012b). However, brain injuries such

Lorenzo et al., 2000). On the other hand, the phosphorylation state as stab lesions and focal ischemia can trigger peripherin expres-

of NFL is regulated by synaptic plasticity events such as long-term sion in CNS neurons normally silent for this gene (Beaulieu et al.,

potentiation (LTP) (Hashimoto et al., 2000b) and long-term poten- 2002). Although deﬁnitive evidence for the presence of periph-

tiation depression (LTD) (Hashimoto et al., 2000a). NFL selectively erin in synapses in the CNS is lacking, electrophysiological studies

inﬂuences dendritic spine and glutamate receptor function since revealed synaptic plasticity was markedly altered in transgenic

NFL-null mice not only have abnormal spines but also dysfunc- mice over-expressing the normal peripherin gene under its own

tion of hippocampal-dependent spatial memory, NMDA receptor promoter (Kriz et al., 2005). LTP was 50% greater in CA3 and 50%

protein expression and synaptic plasticity (Yuan, Veeranna et al. diminished in CA1 of hippocampus of the mutant mice than in wild-

unpublished data). LTP is a persistent increase in synaptic strength type controls. These data indicate a role for peripherin as a mediator

following high-frequency stimulation of a chemical synapse and of plasticity of hippocampal synapses potentially relevant to cogni-

is generally considered one of the major cellular mechanisms that tive diseases. Peripherin was recently shown to be a strong binding

underlie learning and memory (Bliss and Collingridge, 1993). Yuan partner of Rab-7A mutations of which are capable of altering the

A. Yuan, R.A. Nixon / Brain Research Bulletin 126 (2016) 334–346 339

Fig. 2. Model of D1R-containing endosomes anchored on NFM-containing cytoskeletal assemblies. Based on collective ﬁndings on NF scaffolding functions and our D1R data

on NF subunit null mice, we propose a model by which NFM acts in synaptic terminals to anchor D1R-containing endosomes formed after agonist-induced internalization of

membrane D1R. Retention of D1R in a readily available internal pool within the synapse would favor desensitization to D1R stimulation: in the absence of NFM, the greater

recycling back to the plasma membrane surface would favor hypersensitivity to D1R agonists, as observed in our in vivo studies (adapted from Yuan et al. Mol Psychiatry

2016; 20:986-994 with permission).

ratio of soluble and insoluble peripherin in vitro and are causative NR1 receptor and may stabilize its presence in the cell mem-

of CMT2B (Cogli et al., 2013), indicating that peripherin may also brane by preventing its ubiquitination and subsequent degradation

have a role in vesicular transport. More recent studies involving (Ehlers et al., 1998; Ratnam and Teichberg, 2005). Besides bind-

yeast two-hybrid screens established interactions of peripherin ing NR1 to inﬂuence NMDA receptor function, NFL has also been

and SNAP25-interacting protein SIP30 through coiled-coil domains shown to interact in a phosphorylation-dependent manner with

(Gentil et al., 2014) further suggesting a potential important role of 14-3-3 gamma (Miao et al., 2013), another protein implicated in

peripherin in regulating vesicular trafﬁcking at the synapses. schizophrenia (Middleton et al., 2005; Toyooka et al., 1999).

Of further potential relevance to NMDA receptor dysfunction

in schizophrenia is a consistent body of evidence that points to

3. Alterations of NF proteins in psychiatric disorders selectively reduced levels of NFL subunits in brain regions essential

for the cognitive and behavior functions affected in schizophre-

3.1. Schizophrenia and bipolar disorder nia (Kristiansen et al., 2006). In dorsolateral prefrontal cortex

(DLPFC), NFL transcript expression is signiﬁcantly increased across

∼

Schizophrenia, a severe chronic brain disorder affecting 1% all cortical isodense layers in DLPFC from elderly individuals with

of the population (Perala et al., 2007), is characterized by abnor- schizophrenia (mean patient age 80y) while levels of NFL pro-

mal interpretation of reality and social behavior. The behavioral tein are decreased about 50%, collectively suggesting possibly that

syndrome reﬂects a combination of positive symptoms (eg. hal- turnover of NFL is abnormally high. Proteomic analysis of the

lucinations, delusions, and disordered thinking and behavior), DLPFC in schizophrenia revealed synaptic and metabolic abnor-

negative symptoms (eg. reduced expression, feelings, and speech malities, including signiﬁcantly reduced NFL (1.5-fold) and NFM

production), and cognitive deﬁcits (eg. poor executive function- levels (1.2-fold) without changes in NFH and alpha-internexin pro-

ing, working memory, and attention). The causes of schizophrenia teins (Pennington et al., 2008). Reduced NFL and NFM levels were

are unknown. Various alterations of neuronal and glial functions also found in study of DLPFC deep white matter in schizophrenia

have been reported although loss of neurons or neurodegenera- (English et al., 2009) and in the ACC (anterior cingulate cortex)

tive change is not considered fundamental to disease development. white matter (Clark et al., 2007) where a 3-fold increase (p < 0.05)

Postmortem brains from individuals with schizophrenia are usu- in alpha-internexin was also measured. Similar changes in NFL

ally characterized by reduced dendritic spine density (Garey et al., and alpha-internexin levels were seen in another proteomic anal-

1998; Glantz and Lewis, 2000; Sweet et al., 2009). ysis of schizophrenic corpus callosum (Sivagnanasundaram et al.,

Pharmacological and biochemical evidence supports an impor- 2007). Although NFL transcription was signiﬁcantly decreased in

tant role of NMDA receptor hypofunction in the pathophysiology the thalamus of younger adult schizophrenics (mean age 43y), it

of schizophrenia (Balu et al., 2013; Coyle, 2006). Many genetic risk was increased 25%-30% relative to controls, along with NFM levels,

factors for schizophrenia have been reported, including inherited in elderly schizophrenics (mean age 70y) (Clinton and Meador-

common single nucleotide polymorphisms, copy number variants, Woodruff, 2004). Collectively, these results implicate regional

rare single nucleotide variants and rare de novo variants. De novo abnormalities of NFL transcription and/or accelerated NFL degra-

genomic copy number variants known to substantially increase dation in schizophrenia. NFL protein has been shown to be more

susceptibility to schizophrenia are enriched for members of the vulnerable than NFM and NFH subunits to calcium-dependent pro-

NMDA receptor postsynaptic signaling complex and are signiﬁ- teases (Zimmerman and Schlaepfer, 1982). The changes observed

cantly more frequent in individuals with schizophrenia than in in NFs may be part of the dynamic cytoskeletal remodeling in the

controls (Kirov et al., 2012). Fromer et al. conﬁrmed these ﬁnd- pathogenesis of schizophrenia. Dramatic changes in the levels of NF

ings and further implicated abnormalities of synaptic cytoskeleton proteins were also observed in axotomized motor and sensory neu-

regulation and glutamatergic neurotransmission (Fromer et al., rons (Hoffman et al., 1987; Tetzlaff et al., 1996; Wong and Oblinger,

2014). The fact that NFL, NFM and NFH genes map to chromoso- 1990). In light of evidence linking NFL to the functional activity of

mal regions (8p21, 8p22 and 22q12, respectively) that are strongly the NMDA receptor, the lowered levels of NFL could be a poten-

implicated in schizophrenia suggests the involvement of NF pro- tial contributor to the NMDA hypofunction proposed to underlie

teins in this disease (Badner and Gershon, 2002; Lewis et al., 2003). various phenotypic features of schizophrenia.

As earlier noted, NFL in synapses (Yuan et al., 2015b) binds the

340 A. Yuan, R.A. Nixon / Brain Research Bulletin 126 (2016) 334–346

Bipolar disorder is a brain disease that causes extreme mood Rats inbred for alcohol preference expressed lower levels of NF

swings that include emotional highs and lows. The exact cause subunits, suggesting that NF proteins in the VTA may inﬂuence pref-

of bipolar is unknown. Imaging and cellular studies have identi- erence for drugs (Guitart et al., 1992, 1993). Examining its inﬂuence

ﬁed dysfunction of the dorsolateral prefrontal cortex in bipolar on NF proteins, GFAP and NMDA receptors, Ortiz et al. reported that

disease (Drevets et al., 1997; Rajkowska et al., 2001; Strakowski chronic ethanol treatment signiﬁcantly decreased NF protein lev-

et al., 2002). NFL transcripts are reported signiﬁcantly reduced, els in VTA (by 14%-37%) while increasing GFAP (by 40%) and NMDA

along with NFM and alpha-internexin proteins (Pennington et al., NR1 subunit (by 27%) (Ortiz et al., 1995). Similar to other drugs

2008), in adults with bipolar disorder (mean age 42y), but not in of abuse, nicotine activates the mesolimbic pathway by increasing

major depressive disorder (Clinton and Meador-Woodruff, 2004). cell ﬁring of dopaminergic neurons in the VTA via nicotinic recep-

Examining the differential protein expression in the deep white tors (McGehee et al., 1995). Nicotine microinfusions into the VTA

matter from DLPFC from individuals with bipolar disorder and con- which resulted in sensitization of dopamine release (Balfour et al.,

trols, English et al. identiﬁed differences in 15% of proteins classiﬁed 1998), also reduced NFL (by 34%, p < 0.05), NFM (by 42%, p < 0.01),

as cytoskeletal proteins which included increased NFM (1.66 fold, and NFH levels (by 38%, p < 0.05) in the VTA but not in the substantia

p = 0.03)(English et al., 2009). nigra in rats (Bunnemann et al., 2000; Sbarbati et al., 2002) without

Given that most studies of psychiatric disorders focusing on altering neuron numbers. The similar reductions of NF proteins in

cytoskeletal changes analyzed brain areas enriched in white matter the VTA by the chronic treatment of different drugs of abuse with

axons and the alterations of NF proteins may well reﬂect disturbed reinforcing properties suggest that common mechanisms underlie

function of the axonal compartment. Given the ubiquity and abun- these addictive states.

dance of synapses in CNS, it is also possible that even white matter Other brain targets, such as frontal cortex (Busquets et al., 1995;

analyses capture alterations of the signiﬁcant synaptic NF protein Simonato, 1996), also show lowered NFL levels (47%, p < 0.001) in

pool in neuropsychiatric disorders. It is also reasonable to expect brains of addicts dying from opiate overdose (Garcia-Sevilla et al.,

that future deeper analyses that include terminal regions of these 1997). Similar NFL reductions (49%, p < 0.001) are also seen in the

same vulnerable brain regions and target additional relevant grey frontal cortex of rats after chronic morphine treatment. Narayana

matter regions enriched with synaptic boutons, may identify dys- et al. reported immunohistochemical evidence for a signiﬁcant

function of the synaptic cytoskeletal network, including NF subunit decrease in NFH expression in the ﬁmbria and internal capsule of

interactions, more sensitively. rats after chronic cocaine administration (Narayana et al., 2014).

Analysis of NF phosphorylation in the prefrontal cortex of human

opioid addicts uncovered signiﬁcantly reduced nonphosphory-

3.2. Drug addiction lated forms of NFH, NFM, and NFL and corresponding increases

in phosphorylated forms (Ferrer-Alcon et al., 2000, 2003). Similar

Drug addiction is a chronic brain disorder that causes com- changes are seen in brain regions of rats chronically treated with

pulsive drug seeking and use despite adverse consequences. The cocaine (Liu et al., 2003). Acute morphine exposure also induces

mesolimbic pathway, which connects the ventral tegmental area a marked increase in phosphorylated NFH whereas chronic mor-

(VTA) to the nucleus accumbens, has been implicated in common phine administration followed by opiate withdrawal results in a

mechanisms of drug addition (Pierce and Kumaresan, 2006). The time-dependent decline in phosphorylated NFH (Cao et al., 2010;

mesolimbic pathway releases dopamine into the nucleus accum- Jaquet et al., 2001). A recent study found chronic morphine causes

bens, where it affects motivation for rewarding stimuli. Drugs persistent nitration of NFL in cortex and subcortex in mice even

abused by humans such as morphine, cocaine, ethanol and nico- during abstinence (Pal and Das, 2015), although its signiﬁcance

tine have very different chemical structures but all preferentially remains unclear.

increase synaptic dopamine concentrations in the mesolimbic sys- Since PP2A has been shown to be the most effective in the ner-

tem (Di Chiara and Imperato, 1988). This is believed to be the neural vous system in dephosphorylating the sites in NFH known to be

substrate mediating the reinforcing properties of drugs of abuse. phosphorylated by cdk5 (Veeranna et al., 1995), this phosphatase

Given their diverse primary sites of action on cell surface recep- together with cdk5 were also examined. Surprisingly, the levels

tors, these addictive drugs have to act indirectly on intracellular of PP2A were found unchanged while the levels of cdk5 and its

proteins as a convergence point to exert similar effects on mesolim- neuron-speciﬁc activator p35 in the prefrontal cortex of human

bic dopamine function. Beitner-Johnson et al. ﬁrst reported that NF opioid addicts were decreased by 18% and 26–44%, respectively

proteins NFL, NFM and NFH were decreased 15- 50% in the VTA (Ferrer-Alcon et al., 2003). In these brains, phosphorylated NFH

by chronic administration of morphine or cocaine in rats (Beitner- signiﬁcantly correlated with p35 but not with cdk5. Further stud-

Johnson et al., 1992). The lowering of NF protein levels by these ies showed that acute treatment of rats with morphine increased

drugs was selective since the levels of 8 other major cytoskeletal or the density of cdk5 by 35% in the cerebral cortex. In contrast to

cytoskeletal-associated proteins did not change. Chronic exposure the acute effects, chronic morphine induced a marked decrease

to psychotropic drugs lacking reinforcing properties did not alter in cdk5 by 40% and p35 by 47% in rat brain (Ferrer-Alcon et al.,

NF levels. In separate studies, morphine administration caused a 2003). It therefore appears that, despite the possible stimulating

decrease of NFL immunoreactivity in rat cerebral cortex, which was effect of the deadly opiate overdose on cdk5, the reduced expres-

antagonized completely by concurrent administration of naloxone sion of the cdk5/p35 complex in brains of opioid addicts is the

(Boronat et al., 2001). Similar antagonism was achieved in three net result of a chronic opiate effect. These results indicate that

separate knockout mice of opioid receptors (mu or delta or kappa) opioid addiction is associated with down regulation of cdk5/p35

(Garcia-Sevilla et al., 2004), implicating all opioid receptor sub- levels in the brain and the hyperphosphorylation of NFH is not

types in these effects of morphine. Chronic morphine and cocaine the result of a reduced dephosphorylation process. Although the

also increased phosphorylation of NFH and NFM tail domains, an abundance of cdk5 was decreased in the brains from chronic opi-

effect ablated by knockout of cortical mu-opioid receptors (Garcia- oid abusers, Narita et al. showed that the level of phosphorylated

Sevilla et al., 2004). Interestingly, chronic cocaine, but not chronic cdk5 (serine-159) immunoreactivity in the frontal cortex was sig-

morphine, lowered alpha-internexin levels in the VTA, hinting at niﬁcantly increased by chronic morphine treatment (Narita et al.,

somewhat different mechanisms at play for these two drugs, which 2005) and phosphorylation of cdk5 at serine-159 dramatically

are known to act at different receptors (Beitner-Johnson et al., increases cdk5 activation (Sharma et al., 1999). They also demon-

1992). strated that the treatment of selective cdk5 inhibitor, roscovitine

A. Yuan, R.A. Nixon / Brain Research Bulletin 126 (2016) 334–346 341

and a half deletion of the cdk5 gene caused a signiﬁcant inhibition pendently of antibody cross-reaction between NF protein and tau

of the rewarding effect of morphine. In addition, chronic cocaine or (Rudrabhatla et al., 2011). The NFTs are far more complex than that

morphine administration also activates extracellular signal- reg- of tau − PHFs. They also contain abundant NF proteins, vimentin,

ulated kinase (ERK), a known NF protein kinase (Veeranna et al., phosphorylated MAP1, phosphorylated MAP2, nonphosphorylated

1998), by promoting its phosphorylation (Berhow et al., 1996; MAP1B, nonphosphorylated MAP2, nonphosphorylated MAP4 and

Valjent et al., 2000). Altogether, these data indicate the hyperphos- nonphosphorylated MAP6. The question of whether the formation

phorylation of NF proteins under these conditions may be related of tau-PHF is the initiating event in NFT formation has not been

to the up-regulation of phosphorylated cdk5 and increased ERK thoroughly addressed and the possibility that another NFT con-

activity. stituent, like NF protein, may initiate the process has not been

D1R and NMDA NR1 receptors are assembled as an oligomeric excluded. In this regard, Morrison and colleagues reported that, cer-

complex and are functionally interdependent (Fiorentini et al., tain populations of cortico-cortical pyramidal neurons that contain

2003; Lee et al., 2002). Both receptors bind to NF proteins (Ehlers abundant perikaryal NF in relatively low states of phosphorylation

et al., 1998; Kim et al., 2002) and are also involved in drug addic- are highly vulnerable to neurodegeneration through NFT forma-

tion (Comings et al., 1997; Trujillo and Akil, 1991). Cocaine has been tion (Hof et al., 1990; Hof and Morrison, 1990; Morrison et al.,

shown to reduce the D1R-NR1 physical interaction and may inﬂu- 1987; Vickers et al., 1996, 1994). More recent studies have pro-

ence intracellular signaling (Sun et al., 2009). On the one hand, D1R vided independent evidence for loss of SMI32 immunopositive

is a primary target of stimulant drugs and chronic exposure to drugs neurons in temporal cortical areas of AD brain (Thangavel et al.,

of abuse lower levels of NF proteins and alter their phosphoryla- 2009). The loss of SMI32 immunoreactivity on cortical regions of

tion state (Beitner-Johnson et al., 1992). On the other hand, NMDA AD brain is often paralleled by increase in NFTs and AT8-positive

receptor inhibition increases the phosphorylation state and the sol- tau immunoreactivity in neurons (Morrison et al., 1987), indicat-

ubility of NFM protein (Fiumelli et al., 2008). Glutamate has been ing nonphosphorylated NF proteins may play a protective role in

shown to inhibit NF transport, which can be reversed by NMDA preventing the formation of NFTs. In fact, it is suggested that the

receptor blocker MK-801 (Ackerley et al., 2000). Over-activation of development of neuroﬁbrillary pathology may begin with abnor-

primarily NMDA receptors by glutamate leads to rapid disruption mal NF accumulations in damaged distal processes followed by

of NF proteins in cultured neurons (Chung et al., 2005). The basis for reactive perikaryal cytoskeletal changes that ultimately lead to tau

these NF changes and their relationship to synaptic function remain hyperphosphorylation and NFT formation (Vickers et al., 1996). The

elusive. As discussed above, our recent studies demonstrated that co-localization of caspase-3 cleaved spectrin with nonphosphory-

deletion of NFM in mice ampliﬁes D1R-mediated motor responses lated NF-immunoreactive neurons in AD suggests that caspase-3

to cocaine while redistributing postsynaptic D1R from endosomes activation may be a pathological event responsible for the loss of

to the plasma membrane, indicating a role of this NF subunit in drug these vulnerable neurons (Ayala-Grosso et al., 2006).

addiction (Yuan et al., 2015b). NFL mRNA is signiﬁcantly reduced in AD cortex (Clark et al.,

1989; McLachlan et al., 1988) (Kittur et al., 1994) and CA1 and CA2

regions of hippocampus (Somerville et al., 1991). Like NFL, NFM

4. Alterations of NF proteins in Alzheimer’s disease mRNA was also signiﬁcantly decreased while NFH mRNA was unal-

tered in AD temporal cortex as compared to controls (Kittur et al.,

Alzheimer’s disease is an irreversible, degenerative brain dis- 1994). Also signiﬁcantly increased in AD are NFH (by 1.7-fold), NFM

order that progressively destroys memory and other mental (by 1.5-fold) and NFL proteins (by 1.6-fold) and the degree of phos-

functions. Early striking loss of synaptic connections within brain phorylation of NFH (by 1.6-2.7-fold) and NFM (by 1.3-1.9-fold) as

regions involved in memory and thinking skills is followed by loss of compared to Huntington disease (Wang et al., 2001). Proteomic

neurons of many types. Accompanying neurodegenerative changes studies revealed increases of alpha-internexin and NFM proteins

is the development of deﬁning neuropathological hallmarks of AD, in the postsynaptic densities isolated from AD cortex as compared

neuritic plaques, and neuroﬁbrillary tangles, along with extracel- to controls with the increase of alpha-internexin being validated

lular deposition of ß-amyloid. The causes of the most common by Western blots (Zhou et al., 2013). Using quantitative phospho-

late-onset forms of AD (more than 95% of all cases) are not fully proteomic methodology, Pant and colleagues recently reported 13

understood: the much less common early-onset familial forms (less hyperphosphorylated sites in the C-terminal domain of NFM and

than 5% of all cases) are caused by mutations of either of three genes, 10 hyperphosphorylated sites of that of NFH in the frontal cortex

amyloid precursor protein (APP) and presenilins 1 and 2(PSEN1 and of AD brain and all of these sites are in a higher state of phos-

PSEN2) (Hardy and Selkoe, 2002; Van Cauwenberghe et al., 2016). phorylation in AD (4–8-fold higher) compared with control brains

These causative genes and other risk factors inﬂuence the produc- (Rudrabhatla et al., 2010). Surprisingly, the abundance and number

tion and clearance of APP metabolites that mediate APP signaling of KSP repeat hyperphosphorylation of NFM are signiﬁcantly higher

cascades and may also have cytotoxic properties. compared with NFH, even though potential phosphorylation sites

An important feature of AD is the disruption of the neuronal in NFH are more numerous. Based on the NF stoichiometry [4:2:2:1

cytoskeleton leading to formation of neuroﬁbrillary tangles (NFTs) (NFL:alpha-internexin:NFM:NFH)] (Yuan et al., 2006), there are 2-

(Buee et al., 2000; Krstic and Knuesel, 2013) numbers of which fold more KSP repeat sites hyperphosphorylated in NFM compared

correlate well with neurodegeneration and severity of cognitive with NFH, indicating that NFM might contribute more to aberrant

decline in AD (Arriagada et al., 1992; Bierer et al., 1995; Braak NF phosphorylation in AD compared with NFH.

and Braak, 1995). The main constituents of tangles are paired Additional abnormalities of NF have also been observed in

helical ﬁlaments (PHF), initially believed to be composed of NF AD. Signiﬁcantly decreased O-GlcNAcylation of NFM was observed

proteins (Anderton et al., 1982; Dahl et al., 1982; Ihara et al., in AD cortex as compared to controls (Deng et al., 2008) while

1981) and later shown to be mainly ﬁbrillar aggregates of tau pro- carbonyl-related posttranslational modiﬁcation of NFH protein was

tein (Kosik et al., 1986; Ksiezak-Reding et al., 1987; Nukina et al., also present in the AD NFTs (Smith et al., 1995). Chapman et al.

1987). Although NF proteins were somewhat ignored as tangle showed that sera of AD patients contain antibodies that bind specif-

constituents, data has amply conﬁrmed that NF proteins are inte- ically to NFH of Torpedo cholinergic neurons (Chapman et al., 1989).

gral components of the NFTs (Perry et al., 1985). Most recently, AD patients also have elevated intrathecal synthesis of anti-NFH

mass spectrometry analysis by Pant and colleagues conﬁrmed the antibody (Bartos et al., 2012). Behavioral tests revealed that rats

presence of NFM and NFH in puriﬁed NFTs from AD brain inde- immunized with cholinergic NFH for 12 month performed signif-

342 A. Yuan, R.A. Nixon / Brain Research Bulletin 126 (2016) 334–346

icantly worse than controls in T-maze alternation test (Chapman tions beyond caliber expansion. Emerging evidence shows that

et al., 1991). This prolonged immunization of rats with NFH results NF proteins are present and functional in synapses. The synaptic

in cognitive impairment, IgG accumulation in the septum, hip- population of NF proteins is distinctive in phosphorylation state,

pocampus and entorhinal cortex with marked loss of neurons in the likely present in unconventional stoichiometries and assembly

septum (Oron et al., 1997). Interestingly, this cognitive impairment forms including oligomeric structures, and possibly more plastic

can be reversed by the acetylcholinesterase inhibitor physostig- in exchanging subunits and altering form in response to signals.

mine administered 30 min prior to testing (Michaelson et al., 1991). Further evidence that individual subunits differentially modulate

These ﬁndings suggest that NFH may play a role in the pathogenesis neurotransmission and behavior through interactions with speciﬁc

of AD neurons. neurotransmitter receptors heightens expectations that alterations

Further support for NF protein involvement in AD pathogene- of NF proteins could inﬂuence synaptic function in psychiatric dis-

sis comes from genetic studies. Deletion of NFL or NFH subunits in orders and dementias. Despite the very frequent occurrence of NF

T44 mice overexpressing htau resulted in a dramatic decrease in protein changes in multiple major psychiatric states, most of the

the total number of tau-positive spheroids in the spinal cord and analyses have been on white matter tracts and the changes seen,

brainstem, indicating a role of NF proteins in the pathogenesis of often involving levels of only one or two subunit, have been difﬁcult

neuroﬁbrillary tau pathology (Ishihara et al., 2001). Deletion of NFL to interpret mechanistically from a perspective of changes in the

in APP/PS1 transgenic mice, unexpectedly, increased neocortical axonal NF cytoskeleton. The newly uncovered sizable population

ß-amyloid deposition, synapse vulnerability and microgliosis sur- of synaptic NF proteins should encourage more directed attention

rounding ß-amyloid plaques, suggesting a protective role of this NF to regional analyses that interrogate this synaptic NF protein pool

subunit in AD (Fernandez-Martos et al., 2015). and will potentially yield valuable new insights into the signiﬁcance

The abundance of NFs in axons and the frequency of axonal of NF subunits in neuropsychiatric disease.

injury in neurodegenerative disease has led to the use of NF protein

measurements as a clinical index of brain injury in various neuro-

logical disorders. Higher than normal levels of NFL and NFH in CSF Acknowledgements

have been reported in AD (Brettschneider et al., 2006; Hu et al.,

2002) and correlate with disease severity in a recent study of fron-

This work was supported by Grant 5R01AG005604 from the

totemporal dementia (Scherling et al., 2014). CSF NFL concentration

National Institutes on Aging.

is increased by the early clinical stage of AD, correlates with cog-

The authors declare that there are no conﬂicts of interest.

nitive deterioration and structural changes over time (Zetterberg

et al., 2015) and may be a useful marker of disease progression in

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