location, location, location

Location, Location, Location: Altered Transcription FactorTrafficking in Neurodegeneration

Charleen T. Chu, MD, PhD, Edward D. Plowey, MD, PhD, Ying Wang, PhD, Vivek Patel, BA,and Kelly L. Jordan-Sciutto, PhDFrom the Department of Pathology (CTC, EDP), Division of Neuropathology, University of PittsburghSchool of Medicine, Pittsburgh, Pennsylvania; Center for Neuroscience (CTC, VP), University ofPittsburgh, Pittsburgh, Pennsylvania; Pittsburgh Institute for Neurodegenerative Diseases andCenter for the Environmental Basis of Human Disease (CTC), University of Pittsburgh, Pittsburgh,Pennsylvania; and Department of Pathology (YW, KLS-J), University of Pennsylvania, School ofDental Medicine, Philadelphia, Pennsylvania

AbstractNeurons may be particularly sensitive to disruptions in transcription factor trafficking. Survival andinjury signals must traverse dendrites or axons, in addition to soma, to affect nuclear transcriptionalresponses. Transcription factors exhibit continued nucleocytoplasmic shuttling; the predominantlocalization is regulated by binding to anchoring proteins that mask nuclear localization/exportsignals and/or target the factor for degradation. Two functional groups of karyopherins, importinsand exportins, mediate RanGTPase-dependent transport through the nuclear pore. A growing numberof recent studies, in Alzheimer, Parkinson, and Lewy body diseases, amyotrophic lateral sclerosis,and human immunodeficiency virus encephalitis, implicate aberrant cytoplasmic localization oftranscription factors and their regulatory kinases in degenerating neurons. Potential mechanismsinclude impaired nuclear import, enhanced export, suppression of degradation, and sequestration inprotein aggregates or organelles and may reflect unmasking of alternative cytoplasmic functions,both physiologic and pathologic. Some “nuclear” factors also function in mitochondria, and importinsare also involved in axonal protein trafficking. Detrimental consequences of a decreased nuclear tocytoplasmic balance include suppression of neuroprotective transcription mediated by cAMP- andelectrophile/antioxidant-response elements and gain of toxic cytoplasmic effects. Studying thepathophysiologic mechanisms regulating transcription factor localization should facilitate strategiesto bypass deficits and restore adaptive neuroprotective transcriptional responses.

KeywordsAlzheimer disease; Cyclic AMP response element-binding protein; Karyopherins;Neurodegeneration; Oxidative stress; Parkinson disease; Transcription factors

IntroductionWith their highly polarized cell biology, neurons face unique challenges in intracellulartrafficking of key regulatory signaling proteins. Whereas basal transcription of “housekeeping”genes is essential for cell survival, neurons also depend on efficient regulation of transcriptionin response to both physiologic and pathologic stimuli for maintenance of neuronal plasticityand survival (1). At the most basic level, transcription is regulated by the binding oftranscription factors to specific DNA sequences in the promoter region of targeted genes.

Send correspondence and reprint requests to: Charleen T. Chu, MD, PhD, Room W958 BST, 200 Lothrop Street, Pittsburgh, PA 15261;E-mail: [email protected].

NIH Public AccessAuthor ManuscriptJ Neuropathol Exp Neurol. Author manuscript; available in PMC 2008 April 1.

Published in final edited form as:J Neuropathol Exp Neurol. 2007 October ; 66(10): 873–883.

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However, gene expression is also regulated by accessory factors, chromatin modifiers, andsignaling molecules, which couple alterations of gene expressions to physiologic andpathologic stimuli. As DNA is confined to the nucleus (and the mitochondrion, which has itsown system of transcriptional regulation), appropriate subcellular trafficking of transcriptionfactors plays a particularly important role in determining their biologic activities. Recently, agrowing body of literature indicates that some transcription factors display alterations not onlyin expression levels and phosphorylation, but also in their subcellular distribution withinpopulations of neurons affected by neurodegenerative diseases. The implications of alteredtranscription factor localization are not fully understood, although functional repression ofadaptive prosurvival transcriptional programs has been reported (2). In the following sectionwe review the literature on transcription factors and related proteins that display alteredsubcellular localization in human neurodegenerative diseases. This review is followed by adiscussion of nuclear import and export mechanisms with integration of human andexperimental observations to illustrate potential neurodegenerative disease mechanisms andtheir therapeutic implications.

Transcription Factors in the Cytoplasm of Degenerating Human NeuronsSince numerous proteins are alternatively expressed in neurodegenerative diseases such asAlzheimer disease (AD), Parkinson disease (PD), Huntington disease (HD), and amyotrophiclateral sclerosis (ALS) (3), a cohesive conceptual model accounting for these observed changeswould be beneficial. Many proteins and/or their phosphorylated forms that exhibit increasedexpression in neurodegeneration are also alternatively localized with regard to their normallyascribed functions. However, the significance of such changes has often been overlooked orincompletely described. Here we will highlight the importance of subcellular distribution inneurodegenerative diseases by discussing the localization of proteins with well-definedfunctions associated with a specific subcellular compartment—the nucleus (Table). In theinterest of space, we have limited our discussion to a few key transcriptional regulators;however, we hope this review stimulates further interest in the study of how changes insubcellular distribution of proteins may contribute to neurodegenerative disease progression.

Nuclear Factor-κBThe first transcription factor shown to be regulated by altered subcellular distribution wasnuclear factor-κB (NF-κB). The NF-κB family consists of p50, p52, p65, c-Rel, and Rel-B,which share an amino-terminal 300-amino acid Rel homology domain for DNA binding, homo-and heterodimerization and nuclear localization (4). The activity of NF-κB dimers is inhibitedby sequestration in the cytoplasm through interaction with inhibitor of nuclear factor-κB(IκB) proteins, which masks the NF-κB nuclear localization and DNA binding domains. Inneurons, the most common NF-κB complex consists of p65, p50, and IκBα (4).Phosphorylation or oxidation of IκB proteins results in ubiquitination and degradation of IκB.This allows NF-κB to translocate to the nucleus and increase expression of adaptive genes suchas brain-derived neurotrophic factor (BDNF), inducible nitric oxide synthase, and Ca2+/calmodulin-dependent protein kinase II as well as proapoptotic genes such as p53, Fas ligand,and Bcl-Xs (5).

Several stimuli associated with neurodegenerative diseases cause nuclear translocation of NF-κB, including oxidative stress and inflammatory mediators (4), and the distribution of NF-κBhas been investigated in AD, PD, and ALS. In patients with AD, increased levels of neuronalp65 immunoreactivity were observed in the vicinity of early plaques in the basal forebrain,hippocampus, and cortex (6,7). However, active, nuclear p65 immunoreactivity was decreasednear plaques in patients with AD in comparison to age-matched controls (8). In contrast,postmortem PD brains exhibited increased nuclear translocation of NF-κB in dopaminergicneurons of the substantia nigra and ventral tegmental area (9). Neither nuclear nor cytoplasmic

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NF-κB was found to be increased in patients with ALS compared with control patients in 1study (10); however, another report demonstrated nuclear NF-κB in astrocytes of the ALSspinal cord, but not in motor neurons (11). Taken together, these observations suggest thatnuclear translocation of NF-κB differs among several neurodegenerative diseases, which mayprovide clues to the distinct pathology and neuronal subpopulations affected in these diseases.

Activating Transcription Factor 2Activating transcription factor 2 (ATF2) belongs to the basic region leucine zipper family andis an important member of the activator protein-1 (Ap-1) family. ATF2 is ubiquitouslyexpressed in the mammalian brain. Knockout studies revealed that ATF2 is required forneuronal development and migration, but little is known about its specific functions in neuronalcells (12). Localization of ATF2 is regulated by a nuclear export signal (NES) in the leucinezipper region and 2 nuclear localization signals (NLS) in the basic region, resulting incontinuous shuttling between the cytoplasm and the nucleus. Heterodimerization of ATF2 andc-Jun in the nucleus prevents the nuclear export of ATF2 and leads to enhanced transcriptionalactivation (13).

ATF2 expression was investigated in AD, PD, and HD. In AD, there is downregulation ofATF2 expression in the hippocampus (12) and enhanced cytoplasmic ATF2 expression incortical neurons (14). Although these 2 findings are in distinct brain regions, both indicate adecrease in the nuclear/cytoplasmic ratio of ATF2. Many ATF2 target genes are upregulatedin AD. Both c-fos and c-Jun exhibit intense immunolabeling within nuclei in hippocampus inAD brain (15). Interestingly, nuclear localization of the upstream kinases phospho-c-Jun N-terminal kinase (JNK) and phospho-JNK kinase 1 (JKK1) were found only in low-stage AD,suggesting that ATF2 responses are an early event in AD pathogenesis (16). ATF2downregulation has also been observed in the substantia nigra pars compacta (SNc) of PDbrains and in the caudate nucleus of HD brains (12); however, the possibility of early-stageincreases followed by late-stage decreases was not addressed in these studies.

Cyclic AMP Response Element-Binding ProteinCyclic AMP response element-binding protein (CREB) belongs to the family of dimerizingleucine zipper transcription factors, comprising both activating transcription factors such asCREB and activating transcription factor 1 (ATF 1) and repressive transcription factors suchas cAMP-responsive element modulator (CREM) and inducible cAMP early repressor (ICER).As more than 100 genes important for neuron function contain a cAMP response element(CRE) in their promoter region, CREB has become the most widely studied transcription factorin neurons (17) and is implicated in memory, plasticity, and survival (1).

Many signaling pathways converge upon CREB, resulting in phosphorylation of Ser133,including protein kinase A (PKA), calcium-dependent kinases (CaMKs), phosphoinositide-3kinase (PI3K)/Akt, extracellular signal regulated protein kinase (ERK), and phospholipase C(PLC) (18). Phosphorylation at other sites is also believed to modulate activity but has beenless studied.

In cognitively normal populations, immunostaining for CREB is almost exclusively nuclearin the hippocampus, dorsal root ganglia, substantia nigra, and neocortex (19–21). In contrast,brains of patients with AD exhibit decreased levels of the active phosphorylated form of CREB(pCREB) (22). These findings suggest that loss of CREB function may contribute to changesobserved in AD (23). Consistent with this, CREB dephosphorylation has been reported incerebellar granule neurons overexpressing human tau (24). However, further analysis ofpCREB in AD brain tissues is required to determine whether the decrease in pCREB occurs

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through altered expression or phosphorylation and whether subcellular compartmentalizationis affected.

Although the expression of total CREB has not been characterized in the brains of patientswith PD, there are distinct alterations in pCREB localization in degenerating SNc neurons.Nuclear pCREB is not detected in SNc neurons by immunohistochemistry in postmortemhuman tissues but is found under control conditions in cultured primary dopaminergic neurons(2). In PD brains, cytoplasmic aggregates of pCREB are observed in SNc neurons in the absenceof nuclear staining, whereas glial cells in both control and PD patient tissues show nuclearpCREB staining (2). In primary midbrain cultures, 6-hydroxydopamine (6-OHDA) causesprogressive accumulation of pCREB in the cytoplasm and decreased pCREB in the nucleus ofdopaminergic neurons but not in nondopaminergic neurons. Downregulation of CREB-regulated prosurvival gene targets Bcl-2 and BDNF was also observed in 6-OHDA-treatedcells (2). In neonatal 6-OHDA-lesioned rats, nuclear pCREB is also absent in SNc neurons,being restricted to the medial prefrontal cortex (25). As CREB nuclear activities play a centralrole in neuronal adaptation and survival, cytoplasmic sequestration of this transcription factormay represent a novel mechanism contributing to neuronal degeneration and death.

Less is known about the distribution of CREB or pCREB in patient material in HD. However,aberrant transcriptional regulation has been implicated from observations that mutanthuntingtin protein may show DNA binding characteristics. In animal models of HD, it has beenshown that sequestration of CREB binding protein into nuclear aggregates contributes toimpaired CRE transactivation and neuronal cell death (26). Although other studies show anincrease in CRE-reporter transcription in vivo (27), the response element sequences fromdifferent genes may be differentially modulated through cross-talk, and loss of BDNFtranscription has been proposed to play a central role in HD (28). Thus, even when CREB islocalized to the nucleus, adaptive transcription may still be impaired.

TAR DNA Binding Protein 43TAR DNA binding protein 43 (TDP-43) is a highly conserved, widely expressed nuclearribonucleoprotein. It was originally identified by its ability to bind pyrimidine-rich motifs inthe human immunodeficiency virus (HIV)-1 TAR element, functioning as a transcriptionalinhibitor (29). TDP-43 has subsequently been shown to regulate splicing by binding RNA(30); however, its physiologic role remains elusive. Interestingly, TDP-43, which is normallya nuclear protein, was recently reported to accumulate in cytoplasmic inclusions in bothfrontotemporal lobar dementias and ALS (31). Concomitantly, nuclear TDP-43 was reducedin neurons. Furthermore, TDP-43 was universally present in ubiquitin-positive, tau-freeinclusions in all subtypes of frontotemporal lobar dementias, the first protein besides ubiquitinto be localized to tau-free inclusions (31). These observations suggest a role for altered TDP-43function(s) in these neurodegenerative diseases.

p53The transcription factor p53 regulates essential cellular functions including cell cycle arrest,DNA repair, and cell death (32). Mechanisms of p53 regulation are complex; however, itsphysiologic roles are tightly linked to subcellular localization. Nuclear localization of p53enables transcriptional activation of genes that regulate cell cycle arrest and apoptotic cell death(33). Mounting evidence also suggests that p53 can activate apoptosis through a transcription-independent mechanism involving localization to the mitochondria (34).

In postmortem brain tissues from patients with AD (35), diffuse Lewy body disease,frontotemporal lobar dementia, PD, and ALS, increased p53 immunoreactivity was observedin neuronal nuclei coincident with nuclear DNA fragmentation (36). However, increased p53

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in other neurons was sometimes diffuse or cytoplasmic (36). Finding nuclear p53 in conjunctionwith DNA fragmentation (37) is consistent with the well-characterized transcription-dependentapoptotic pathway induced by p53 in numerous non-neuronal cells. However, the localizationpattern in neurons without DNA fragmentation needs to be considered further in comparisonwith controls to understand the significance of increased cytoplasmic p53 (34,38). The role ofp53-dependent cell death in experimental models of neurodegeneration is highly controversial.There is as much evidence to support a role for p53 in neuronal cell death as there is evidencethat p53 is not required. The significance of p53 in neurodegenerative disease needs to beconsidered in light of its subcellular distribution and the mechanisms that regulate thesechanges.

Sma/Mothers Against DecapentaplegicThe transcription factors that function downstream of transforming growth factor β can bedivided into receptor-regulated Sma/mothers against decapentaplegic (Smads), which interactwith Smad 4 to transduce signals, and the inhibitory decoy Smads (39). Nuclear retention ofRSmads is stabilized by phosphorylation-dependent binding to Smad 4, allowing recruitmentof DNA-binding cofactors. Smad 2 enters the nucleus via direct nucleoporin interactions,whereas both direct FG interactions and importin-dependent pathways are important for Smads3 and 4. Interestingly, a recent study shows decreased Smad 3 nuclear localization in tangle-bearing neurons in AD and increased cytoplasmic Smads associated with insoluble tau andgranulovacuolar degeneration (40).

E2 Promoter Binding Factor 1E2 promoter binding factor (E2F) transcription factors participate in cell cycle progression andapoptosis in non-neuronal cells (41) and have been linked to regulation of neuronal survivaland death (42). The ability of E2F1 to promote cell cycle progression or apoptosis depends ontranscriptional activation of a subset of E2F target genes. Transactivation by E2F1 is repressedby interaction with the retinoblastoma protein in most cell types (43); however, in neurons, anovel, melanoma antigen gene (MAGE)-like protein, necdin, has also been linked to repressionof E2F1 activity (44). Endogenous E2F1 protein levels are nearly undetectable in postmitoticneurons, but E2F1 mRNA is abundantly expressed (45,46), so the low level of E2F1 proteinmay contribute to maintenance of the postmitotic phenotype in neurons. E2F1 has a NLS andDNA binding activity. Moreover, treatment of primary cortical neurons with an inhibitor ofnuclear export, leptomycin B, led to nuclear accumulation of E2F1, whereas untreated cellshad predominantly cytoplasmic E2F1 (47). This suggests a role for active nuclear export,although the E2F1 NES remains undefined (48).

Whereas mature neurons permanently exit the cell cycle, S-phase reentry has been implicatedin neurodegeneration (46). As E2F1 is principally responsible for S-phase progression, itsexpression in neurodegenerative diseases was explored in AD, ALS, and HIV encephalitis. InAD and ALS, E2F1 was observed predominately in the cytoplasm of cortical neurons andmotor neurons of the spinal cord, respectively (49–51). In another neurodegenerative disease,HIV encephalitis, E2F1 protein was increased in neurons and glia in both the nucleus andcytoplasm (52–54); however, potential alterations in the nuclear/cytoplasmic ratio were notaddressed in these studies. The presence of E2F1 in the cytoplasm has only been reported in 1other cell type, mature skeletal myocytes, another terminally differentiated cell population(55). The role of cytoplasmic E2F1 is not known, although it has been reported that expressionof E2F1 causes neuronal degeneration coincident with aberrant S-phase entry (56), and E2F1can induce apoptosis independent of its transcriptional activity (57). In non-neuronal cells,E2F1 mutants lacking the NLS and DNA binding domain induce calpain activation, and asignificant reduction in antiapoptotic MDMX protein was coincident with an increase in E2F1in a simian model of HIV encephalitis (47). Additional studies are needed to elucidate the

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function(s) of cytoplasmic E2F1 and mechanisms by which the balance of nuclear versuscytoplasmic E2F1 may regulate neuronal survival.

Nuclear Factor E2-Related Factor 2Nuclear factor E2-related factor 2 (Nrf2) is a member of the Cap ‘n’ Collar family of basicleucine zipper transcription factors, which includes Nrf1, Nrf3, p45, Bach1, and Bach2 (58).Nrf2 appears to be a master regulator of the cellular response to electrophilic xenobiotics andoxidative stress. Promoter regions of protective genes, such as those encoding phase IIdetoxification enzymes, the antioxidant copper zinc superoxide dismutase, and enzymesubunits involved in glutathione synthesis contain the DNA binding site for Nrf2, which isknown as the electrophile response element (EpRE) or antioxidant response element.

Nrf2 activity is predominantly regulated by subcellular distribution (59), as Nrf2 mRNA levelsremain relatively constant in response to oxidative stressors (60). In unstressed cells, Nrf2 isretained in the cytoplasm by the actin-binding, cytoplasmic Kelch-like ECH-associated protein1 (Keap1) and ubiquitinated by the Cul3 ligase leading to proteasome-mediated Nrf2degradation (60). Disruption of the Keap1:Nrf2 interaction by phosphorylation of either proteinor by oxidation of Keap1 sulfhydryl residues results in nuclear import of Nrf2 andtranscriptional activation (61). A variety of kinases have been implicated in phosphorylationof Nrf2 including PKC, type I transmembrane endoplasmic reticulum-resident protein kinase(PERK), PI3K, and ERK/mitogen-activated protein kinase (MAPK) (62).

Oxidative stress has been implicated in the pathologic mechanisms of many neurodegenerativediseases, including AD, PD, and ALS. Whereas Nrf2 normally shows a nuclear distribution inthe hippocampus, in AD and the Lewy body variant of AD, Nrf2 accumulated in the cytoplasmof hippocampal neurons (63). In contrast, localization of Nrf2 was nuclear in SNc neurons ofPD. Further investigation of Nrf2 targeted gene expression in neurodegenerative diseases andthe potential effects of premortem drug therapy will aid in understanding the differences in theresponse of neuronal Nrf2 in these patient populations. For example, deprenyl treatmentinduces Nrf2 nuclear localization and EpRE-mediated gene expression in SH-SY5Y cells(64), and Nrf2 responses can protect against 6-OHDA toxicity in vitro and in vivo (65,66).These findings suggest that altered localization and activity of Nrf2 may contribute toneurodegeneration and may be an important target for therapeutic intervention.

Cytoplasmic Retention of Activated Transcriptional Regulators: PotentialMechanisms

Given that signaling proteins and transcriptional factors are thought to exhibit dynamicshuttling between nuclear and cytoplasmic compartments, with shifts in the predominantdistribution triggered in response to stimuli, there are several potential mechanisms that mightresult in the perturbed distribution of transcription factors observed during neurodegeneration(Fig. 1). These include compartmentalized cytoplasmic or organelle-associated activation,altered levels of or access to compartment-specific anchoring proteins, impaired nuclearimport, enhanced nuclear export, inefficient degradation/dephosphorylation, and/orsequestration in protein aggregates.

Regulation of Classic Nuclear Import and ExportNuclear transport of proteins has been extensively reviewed (67–69). Briefly, nuclear transportis mediated by macromolecular nuclear pore complexes composed of nucleoporins with FGrepeats that are important in pore selectivity. Whereas small proteins <40 kDa and metabolitescan passively equilibrate across the nuclear envelope, karyopherin proteins (importins andexportins) mediate active transport into and out of the nucleus (Fig. 1A). Classical import

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involves recognition of cargo bearing an NLS by the adapter importin α. The importin alpha-cargo complex is escorted through the NPC by importin β via interactions with nucleoporinFG repeat motifs. Whereas importin β can directly bind some cargo proteins, recruitment ofan array of adaptors and heterodimerization with other importin β-like receptors greatly expandthe cargo repertoire.

The small G protein Ran plays a critical role in both import and export cycles. RanGTP bindsto importin β on the nuclear side of the NPC to induce dissociation of the complex, releasingthe cargo protein into the nucleus. RanGTP-bound importins are then returned to the cytoplasmand released upon GTP hydrolysis. Nuclear export is mediated by exportins such as Crm1,which chaperone cargo displaying NESs in complex with RanGTP through the NPC. Cargorelease to the cytoplasm occurs upon stimulation of RanGTP hydrolysis by RanGAP. Ran itselfis returned to the nucleus by nuclear transport factor 2/p10, where it is reloaded with GTP withthe assistance of a Ran guanine nucleotide exchange factor (RanGEF) located inside the nuclearenvelope and released to maintain a steep nuclear-to-cytoplasmic RanGTP gradient.

Importins and Axonal TransportIn addition to mediating nuclear import, there are several other functions of importin β familymembers that may be relevant to neurodegeneration. First, importin β heterodimers canfunction as cytoplasmic chaperones, shielding highly basic proteins and preventing proteinaggregation (67). Secondly, importins have been shown to play an important role in injury-induced axonal transport along microtubules (69). Injury induces localized axonal translationof importin β, which shepherds cargo along microtubes in a dynein-dependent mannerinvolving importin α and intermediate filaments (70). Excess NLS peptides introduced to theaxon competes for importin-mediated trafficking and significantly delays regenerativetranscriptional responses. These studies emphasize the critical role of well-regulated traffickingof transcriptional regulators, often over long distances, for successful adaptation of the neuronto physiologic (plasticity) and pathologic (injury or disease-associated) stimuli. Impairment ofadaptive signal transmission, as observed in models of oxidative neuronal injury, couldcontribute to loss of functional viability in the setting of chronic neurodegenerative diseases.

Localized Activation and Alterations in Anchor ProteinsMany signaling proteins and transcription factors are maintained in the cytoplasm throughbinding interactions with cytoplasmic anchors, which are normally released upon initiation ofsignaling cascades that promote transcription. For example, the interaction of the transcriptionfactor Nrf2 with its cytoplasmic anchor Keap is disrupted by phosphorylation or cysteineoxidation of Keap, allowing nuclear translocation of Nrf2 to promote EpRE-mediatedtranscription (62). Similarly, the interaction of NF-κB with its anchor IκB is released uponIκB phosphorylation or oxidation. As variations on this theme, phosphorylation of thetranslocating kinase can cause its release from cytoplasmic anchors, as observed for ERKsignaling, and, conversely, dephosphorylation of Forkhead box transcription factor O unmasksits NLS to promote nuclear import (71). Any process that results in increased binding tocytoplasmic anchors, such as oxidative cross-linking, protein aggregation, or increased anchorprotein expression, could serve to tip the nuclear to cytoplasmic balance of these dynamicsignaling proteins.

Association of activated signaling proteins and transcriptions factors with cytoplasmicorganelles may represent another mechanism for increased cytoplasmic localization. Thegrowing field of mitochondrial kinase and transcription factor regulation has been the subjectof a recent review (72). For example, CREB and its activating kinase PKA are localized to themitochondrial matrix (73) and function to regulate both nuclear and mitochondrialtranscription. Likewise, mitochondrial reactive oxygen species mediate delayed redox

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activation of mitochondrial ERK in a culture model of PD (74). Additional studies of organelle-targeted signaling and activation of other scaffolded signaling complexes are likely to revealnovel physiologic as well as pathologic functions for signaling proteins and transcriptionfactors in cytoplasmic compartments.

On the other hand, interactions with chromatin or the nuclear scaffold delay rates of nuclearexport, and a few transcriptional regulators are maintained in the nucleus through interactionwith anchors such as dTCF/Pangolin for Armadillo and phosphatases for ERK (75,76), whichfunction to regulate duration of ERK activity and its localization. Loss, inaccessibility, ordecreased affinity for these binding sites may facilitate a decreased nuclear to cytoplasmicbalance and cytoplasmic accumulation of transcription factors.

Nuclear Transport Impairment With Oxidative StressThe effects of oxidative stress on nuclear import mechanisms in non-neural cell culture systemshave been examined in a few studies. Hydrogen peroxide induced a dose-dependentimpairment of nuclear import in permeabilized aortic smooth muscle cells (77). Subsequentstudies in HeLa cells (78) demonstrated that hydrogen peroxide treatment resulted indissipation of the nucleocytosolic Ran-GTP gradient and redistribution of nucleoporin Nup153immunofluorescence from the nuclear periphery to throughout the nucleus. Degradation ofNup153 and other components of the nuclear transport machinery was demonstrated andpartially reversed by caspase inhibitors (78). Dissipation of the Ran gradient by hydrogenperoxide was also associated with impaired nuclear export of importin α (79), which wouldresult in a nuclear import deficit due to impaired recycling of limiting amounts of importins.Taken together, these findings may relate to apparent derangements in nuclear localization ofsignaling proteins in models of neurodegeneration. Although the results of Ran-relateddysfunction on the localization of specific transcription factors are difficult to predict, as importand export cycles are so closely tied, it is possible that specific components of the nucleartransport machinery are sensitive to redox regulation. Depending on the extent of redox stress,there may be selective perturbations in certain signaling and transcriptional pathways thatimpair the neuron's ability to adapt to stresses. More severe levels of damage may then lead towidespread impairment of cellular trafficking and signaling pathways.

Another potential mechanism of impaired nuclear import may involve modifications of NLSproteins or of importins, either of which could inhibit cargo recognition. As phosphorylationof transcription factors near the basic NLS can decrease binding to importin α, oxidative ornitrosative additions may have similar effects. As there are at least 10 human importins, subsetsof nuclear cargo might be affected by redox modulation of specific importins. Even if thetranscription factor is successfully imported, modifications such as nitroalkylation of NF-κBcan lead to decreased DNA binding and decreased transcriptional activity (80). The loss of theanchoring property of DNA interactions could also enhance nuclear export of activatedtranscription factors, contributing to increased steady-state concentrations of cytoplasmictranscription factors.

It is possible that cytoplasmic distribution reflects passive leakage secondary to nuclearenvelope damage. Structural alterations of the nuclear envelope have been described in tissuesof AD patients (81). Gross disruption of the nuclear envelope, as that occurring duringhyperacute cell death or in the late stages of apoptosis, could result in leakage of nuclearproteins due to passive equilibration of soluble proteins. However, there does seem to beselectivity in the spectrum of nuclear proteins observed accumulating in the cytoplasm duringoxidative neuronal injury and neurodegeneration. Thus, it is likely that more selectivedisruptions in nucleocytoplasmic shuttling underlie the specific alterations observed in bothearly and advanced stages of chronic neurodegenerative diseases.

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Protein Aggregation and Altered Degradation/Dephosphorylation MechanismsThe characteristic pathologic hallmarks of most neurodegenerative diseases include some formof ubiquitinated protein aggregates. Interestingly, abnormal accumulation of importin α hasbeen observed in neuronal Hirano bodies in AD, suggesting the possibility of functionalimpairment (82). Tangles and granulovacuolar inclusions have been implicated in sequesteringand preventing nuclear localization of pSmad 3 in AD brains (40). Moreover, phosphorylatedCREB kinases are observed in the halo region of Lewy bodies and in association with abnormalα-synuclein deposits (83). The transcription factor Elk-1 has also been observed in glialcytoplasmic inclusions of multiple system atrophy (84), another neurodegenerative diseasecharacterized by α-synuclein aggregation. In experimental systems, overexpression of α-synuclein drives robust ERK phosphorylation, aberrant expression of cell cycle proteins (85),and sequestration of the transcription factor Elk-1 in cytoplasmic aggregates (84). The PD toxinMPP+ causes increased levels of both α-synuclein and phosphorylated ERK (86), suggestingcommon mechanisms between genetic and toxin models of PD. Finally, sequestration of CREBbinding protein into nuclear aggregates has been shown to contribute to impaired CREtransactivation and neuronal cell death in models of polyglutamine repeat diseases (26). Thus,even when proteins traffic correctly to the nucleus, adaptive transcription may still be impaired.

In addition to nuclear import, there are other prominent mechanisms that limit the lifetime ofactivated signaling proteins in the cytoplasm. These mechanisms include dephosphorylationand degradation. Thus, it is possible that accumulation of activated signaling proteins in thecytoplasm reflects dysfunction of phosphatases, which can be sensitive to oxidativeinactivation (87). Alternatively, the phosphorylated transcriptional regulators may beinaccessible to phosphatases, due perhaps to sequestration in aggregates or in autophagicvacuoles. Cytoplasmic anchoring systems for several transcription factors, such as Nrf2 andNF-κB, often function to also target the factor for proteasomal degradation. Impairment ofproteasome function is a feature of many neurodegenerative diseases (88), andmacroautophagy may be induced as a backup clearance mechanism or in the context ofautophagic cell death (89,90). It is possible that some mislocalized proteins are merely passivemarkers of an underlying neurobiologic dysfunction that mediates neurodegeneration. Studiesin experimental models of several neurodegenerative diseases, however, suggest that thefunctional consequences of altered transcription factor localization contribute actively toneurodegeneration and neuronal cell death.

Transcription Factor Localization in Neurodegeneration: TherapeuticImplications

The potential functional consequences of nonclassic transcription factor trafficking anddistribution are numerous. One potential explanation for observations in neurodegenerativediseases is that the protein is normally alternatively localized in neurons compared with non-neuronal cells, based on distinct neuronal import and export machinery or distinct regulatorymechanisms for a given transcription factor in neuronal subpopulations. For example, E2F1interacts with the neuron-specific protein, necdin (44) and exhibits distinct localization inprimary neurons in vitro (91). If the protein is truly altered in its localization in subsets ofneurons undergoing neurodegeneration compared with control conditions, these changes mayaffect neuronal function and activity in different ways. By not localizing to the nucleus,transcription factor-mediated regulation of neuronal gene expression may be lost. As many ofthese signaling pathways regulate neuronal survival, altered localization may contribute to celldeath commitment. Loss of nuclear localization of other transcriptional regulators may alsoserve to decrease expression of gene products needed for synaptic maintenance, leading tofunctional disturbances and neurite degeneration. Alternatively, the protein may gain aberrantfunctions from prolonged activation in the cytoplasmic compartment. Finally, divergent roles

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for signaling proteins and transcription factors may be unmasked under conditions of stress ordisease. A protein may be diverted from its usual role in neurons to alternative roles as part ofa stress response. In the short term, this adaptive response may be tolerated; however, duringchronic stress of neurodegenerative disease, loss of normal function may ultimatelycompromise survival.

Nongenomic Functions of Transcription Factors and Their Upstream RegulatorsIn addition to cytoplasmic retention of transcription factors, upstream regulatory kinases oftenshow increased expression or altered localization in neurodegenerative diseases. In particular,mitogen-activated protein kinases act upstream of ATF2, Elk, CREB, and Nrf2. Increasedactivation of JNK, ERK, p38 MAPK, JKK 1, MAPK/ERK kinase 1 (MEK 1), and MAPKkinase kinase 6 (MKK6) have been reported in AD hippocampus (16,92,93) and in themidbrains of patients with PD, Lewy body dementia, and progressive supranuclear palsy (83,94). Ultrastructural and dual fluorescence studies indicate distribution of phospho-ERK infibrillar bundles, mitochondria, and autophagosomes (95). Furthermore, mitochondrialoxidative stress may be involved in localized redox activation of ERK (74).

Potential cellular effects of cytoplasmic ERK activation include regulation of autophagy (96),calpain activation (97), mitochondrial alterations (98,99), and, interestingly, inhibition ofnuclear import in hydrogen peroxide-treated cells (77). The potential roles of autophagy,calpains, and abortive cell cycle reentry in neurodegeneration have been reviewed elsewhere(46,90,100,101). Other mitochondrial signaling pathways include the PKA/CREB system, inwhich localization is mediated by mitochondrial A-kinase anchoring proteins. Interestingly,pCREB is found in human neuronal mitochondria, and culture studies indicate that CREB isinvolved in modulating mitochondrial transcription and prosurvival functions (73). Thus,cytoplasmic localization of certain transcription factors may reflect normal, rather thanpathologic, functions yet to be elucidated.

Loss of Neuroprotective Transcriptional ResponsesIn addition to pathologic gain-of-function effects attributed to sustained activation of signalingproteins in the cytoplasm, impairment of nuclear import would be expected to block prosurvivaltranscriptional responses by activated signaling proteins. CREB is a downstreamtranscriptional effector of ERK (102), which exemplifies this mechanism (2). CREB trafficsto the nucleus in an importin β- and RanGTP-dependent mechanism (103), where it isphosphorylated. In contrast, cytoplasmic accumulations of pCREB are detected in primarycultures and cell lines exposed to 6-OHDA, as well as in human PD substantia nigra neurons(2). 6-OHDA decreases the expression of CRE-containing prosurvival genes BDNF and Bcl-2(2), but treatment of cells with cAMP after injury restores CRE transactivation and confersprotection (2). Interestingly, kinase-induced CRE activation requires active nuclear import, butcAMP-induced CRE activity does not (104), suggesting a mechanism to bypass deficits inactive import. Alternatively, it is possible that cAMP reverses the nuclear import impairment,allowing primed cytoplasmic pCREB to regulate transcription. Regardless of the precisemechanism, these data provide proof of concept information that strategies to bypass or reversemechanisms leading to altered nucleocytoplasmic shuttling of transcription factors canalleviate signaling dysfunction in oxidatively stressed neurons, conferring neuroprotection.

Unresolved Issues and Therapeutic ImplicationsSubcellular distribution of signaling proteins is important for normal cellular function andefficient responses to cellular stress. Furthermore, it is emerging as an important regulatorymechanism that is perturbed during the progression of neurodegenerative disease. Proteinslocalized to compartments that are distinct from their better-known functions are heterogeneousin their activities, roles, and regulatory mechanisms. This heterogeneity may account for the

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recalcitrance of neurodegenerative diseases to single modality therapies. Most importantly, thereasons for alternative localization are still not understood for most of these proteins. To devisemechanistically driven treatments for these diseases, it will be necessary to understand thesignificance of altered localization of a given group of proteins, the mechanisms accountingfor altered localization, and the best way to modulate and restore appropriate localization andfunction. For example, simple trophic factor replacement therapies may be destined to fail ifpotential neurodegenerative impairments in downstream signaling to the nucleus are notconsidered. Future researchers should not only address altered expression levels of proteins inneurodegenerative disease but also delineate subcellular compartments of activation and thecell types impacted to yield a more complete picture of signaling dysfunction inneurodegenerative diseases.

Acknowledgements

We thank Dr. Donald DeFranco of the University of Pittsburgh for helpful discussion.

The authors' research studies on signaling in neurodegeneration have been supported by funding from the NationalInstitutes of Health (Grants NS040817, NS053777, NS41202, and AG026389) and by the Pittsburgh Foundation JohnF. and Nancy A. Emmerling Fund.

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FIGURE 1.(A) Schematic diagram of a generalized signal transduction pathway resulting in activationand nuclear import of a transcription factor and subsequent transcriptional activation. Underresting conditions (1), the transcription factor is bound to a cytoplasmic anchoring protein.Receptor ligation and/or physiologic redox signals result in phosphorylation of the transcriptionfactor and/or phosphorylation or oxidation of the anchor and release of the transcription factor(2). The exposed nuclear localization signal (NLS) is recognized by importins, which escortthe transcription factor into the nucleus (3). Importin β is also involved in retrograde transportof activated signaling proteins along neuritic processes (4). Within the nucleus, RanGTP bindsimportin β, which promotes disassembly of the import complex (5), freeing the transcription

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factor to bind DNA and form active transcription regulatory complexes. Transcription may beterminated by nuclear phosphatases and/or proteasomal degradation (6). Nuclear export ismediated by complexes consisting of cargo, an exportin, and RanGTP. Near the cytoplasmicface of the nuclear envelope, Ran GTPase-activating protein (RanGAP) maintains the nuclear-to-cytoplasmic RanGTP gradient by catalyzing GTP hydrolysis (7), promoting disassembly ofthe export complex. Importins are also recycled to the cytoplasm through a RanGTP-mediatedmechanism. (B) Proposed mechanisms through which oxidative stress could impair nucleartransport of transcription factors, depriving the cell of adaptive, prosurvival transcriptionalsupport and eliciting potentially detrimental cytosolic effects. Under conditions of oxidativestress such as those that occur during acute and chronic neurodegenerative insults, adaptivesignaling pathways are activated by phosphorylation and/or redox mechanisms. However,post-translational modifications may impair cargo recognition by importins (1). Oxidativecross-linking of proteins, the presence of mutant neurodegenerative disease-associatedproteins, and/or oxidative inactivation of phosphatase or proteasomal function could promoteprotein aggregation, resulting in further sequestration of signaling proteins (2). Sustainedcytoplasmic signaling and/or organelle-targeted activation could further contribute topathogenic mechanisms, such as calpain activation, altered mitochondrial function orautophagic stress from dysregulated autophagy (3). Alternatively, the nuclear pore complexitself could be the target of oxidative damage, with impairment of import and/or leakage ofproteins from the nucleus (4). Intranuclear aggregation or other post-translational or oxidative/nitrosative modifications may further interfere with proper transcriptional responses (5),resulting in loss of stabilizing transcription complex-DNA interactions. Dissipation of theRanGTP gradient and/or impairment of importin recycling could also affect nuclear trafficking(6). Finally, lack of transcriptional support and/or oxidative or aggregative damage to axonsand dendrites could result in further impairment in transmission of signals needed formaintenance and/or repair of neurites and synaptic structures (7). ROS, reactive oxygenspecies.

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TABLESummary of Nuclear Import-Export Mechanisms for Shuttling Transcriptional Regulators

Protein Regulation of Nucleocytoplasic Shuttling Import-Export Signals

NF-κB Phosphorylation or oxidation of its cytoplasmic anchor IκB results in itsubiquitination and degradation, releasing NF-κB to undergo nuclear import

Active mechanisms involving NLS andNES

CREB Classic mechanism involves phosphorylation of the nuclear pool of CREB. Kinaseand depolarization stimuli elicit cytoplasmic CREB phosphorylation followed byRan-dependent import

Active mechanism involving NLS

TDP-43 Unknown except that neurons with ubiquitinated inclusions show loss of nuclearstaining

Unknown

p53 p53 can be sequestered in cytoplasm by association with a vimentin scaffold. TheNES is masked by tetramerization of p53; enhanced export and degradation ismediated by association with MDM2

Active mechanism involving NLS andNES

ATF2 Monomers shuttle freely, but heterodimerization with activated c-Jun stabilizeslocalization in nucleus


E2F1 Normal location is nuclear; no classic NES, but undergoes calcium-induced activeexport and degradation

Active mechanism involving NLS; exportsignal unknown

Smad Undergoes constitutive nucleocytoplasmic shuttling; phosphorylation of Smad 2/3allows binding to Smad 4 to form active complex that accumulates in nucleus

Import and export by direct interactionwith nucleoporin FG repeats; Smads 3and 4 also utilize NLS-dependent import

Nrf2 Phosphorylation or oxidation of cytoplasmic anchor Keap1 results in dissociationand nuclear import of Nrf2


ERK ERK phosphorylation results in dissociation from cytoplasmic anchor MEK,allowing ERK import by multiple mechanisms. Export is mediated by binding toNES-containing proteins such as MEK or the phosphatase MKP3, whereas DUSP5functions as a nuclear anchor

Multiple mechanisms involving protein-protein interactions including direct NPCFG repeat interactions, diffusion, andRan-dependent import

See text for references. NF-κB, nuclear factor-κB; IκB, inhibitor of nuclear factor-κB; NLS, nuclear localization signal; NES, nuclear export signal; CREB,cAMP response element-binding protein; TDP-43, TAR DNA binding protein 43; ATF2, activating transcription factor 2; E2F1, E2 promoter bindingfactor 1; Smad, Sma/mothers against decapentaplegic; Nrf2, nuclear factor E2-related factor 2; ERK, extracellular signal-regulated kinase; MEK, mitogen-activated protein kinase kinase; NPC, nuclear pore complex; MKP3, mitogen-activated protein kinase phosphatase 3; DUSP3, dual specificity phosphatase3 (vaccinia virus phosphatase VH1-related).


location, location, location

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