Wld S but not Nmnat1 protects dopaminergic neurites from MPP+neurotoxicity
© Antenor-Dorsey and O'Malley; licensee BioMed Central Ltd. 2012
Received: 10 August 2011
Accepted: 8 February 2012
Published: 8 February 2012
The Wld S mouse mutant ("Wallerian degeneration-slow") delays axonal degeneration in a variety of disorders including in vivo models of Parkinson's disease. The mechanisms underlying Wld S -mediated axonal protection are unclear, although many studies have attributed Wld S neuroprotection to the NAD+-synthesizing Nmnat1 portion of the fusion protein. Here, we used dissociated dopaminergic cultures to test the hypothesis that catalytically active Nmnat1 protects dopaminergic neurons from toxin-mediated axonal injury.
Using mutant mice and lentiviral transduction of dopaminergic neurons, the present findings demonstrate that Wld S but not Nmnat1, Nmnat3, or cytoplasmically-targeted Nmnat1 protects dopamine axons from the parkinsonian mimetic N-methyl-4-phenylpyridinium (MPP+). Moreover, NAD+ synthesis is not required since enzymatically-inactive Wld S still protects. In addition, NAD+ by itself is axonally protective and together with Wld S is additive in the MPP+ model.
Our data suggest that NAD+ and Wld S act through separate and possibly parallel mechanisms to protect dopamine axons. As MPP+ is thought to impair mitochondrial function, these results suggest that Wld S might be involved in preserving mitochondrial health or maintaining cellular metabolism.
Parkinson's disease (PD) is the second most common neurodegenerative disorder in the U.S., affecting 1-2% of people over the age of 55. Characterized by loss of dopaminergic neurons in the substantia nigra (SN) [1, 2], the cardinal motor symptoms of PD include resting tremor, bradykinesia, rigidity, and abnormal gait [3, 4]. Another characteristic of PD is its late onset and progressive nature. Symptoms appear after 50-70% [5, 6] of striatal dopamine has been depleted and 30-50% [7, 8] of the nigral dopaminergic cells have died. Such studies suggest that the extent of striatal dopamine depletion is better correlated with the severity of PD symptoms than the loss of dopaminergic neurons in the SN .
Data from PD-linked genetic mutations also support the notion that axonal pathology and/or dysfunction occurs prior to the loss of dopaminergic cell bodies. For example, α-synuclein pathology is seen in neurites before it is observed in PD-associated cell bodies [3, 9]. α-synuclein mutants accumulate in the cell soma when overexpressed in cortical neurons, suggesting impaired axonal transport as well . Moreover, transgenic models expressing the PD-linked mutant gene leucine rich repeat kinase 2 (LRRK2) also exhibit pronounced axonal loss and pathology prior to cell body loss . In addition, genetic mutations in other PD-linked genes such as Parkin, an E3 ligase , and PINK1 (PTEN-induced putative kinase 1 protein) a mitochondrially-targeted kinase, also alter axonal transport [13, 14]. Collectively, these findings have led to the idea that nigral neurons degenerate through a "dying back" axonopathy where degeneration starts in the distal axon and proceeds over time towards the cell body.
Environmental toxins known to mimic PD such as rotenone and MPP+ also disrupt axonal function. These factors not only inhibit mitochondrial Complex I activity, but also de-polymerize microtubules leading to axon fragmentation and decreased synaptic function [15–17]. Moreover, MPP+ can directly inhibit axon transport in the squid axoplasm  and DA neurons . Thus, results from PD-associated environmental and genetic factors support an early, critical role for axonal impairment in PD.
Recent data suggest that the Wallerian degeneration slow fusion protein (Wld S ) can delay axonal degeneration about 10-fold from a wide variety of genetic and toxin-inducing stimuli in the peripheral nervous system . Wld S also blocks axon degeneration in several central nervous system (CNS) models of degeneration including animal models of PD [21, 22]. For example, we previously found that Wld S rescues 85% of dopaminergic axons for at least 7 days post MPTP treatment in vivo . Because no other mutation or drug protects axons as robustly as Wld S , understanding how the Wld S fusion protein is able to prevent axon degeneration is the first step towards defining an intervention that would leave axons intact.
Wld S is a chimeric protein composed of the first 70 amino acids of the ubiquitination factor E4b (Ube4b) followed by an 18-amino acid linker region and then the entire coding sequence for nicotinamide mononucleotide adenylyltransferase (Nmnat1), a nicotinamide adenine dinucleotide (NAD+) synthesizing enzyme [24, 25]. Most studies suggest that catalytically active Nmnat1 is necessary for axonal protection [26, 27], hence, exogenous addition of NAD+ has been reported to delay Wallerian degeneration in response to axotomy in dorsal root ganglion (DRG) cells . In Drosophila, however, the picture is more complex in that Avery et al. , showed that Nmnat enzymatic activity is required following axotomy whereas Zhai et al.  found that Nmnat does not need its catalytic domain to protect axons. In this model , as well as in a new study demonstrating that Nmnat also protects dendrites , Nmnat exhibits a separate chaperone-like activity which protects axons and dendrites [30, 32].
Inasmuch as most studies have been done in peripheral model systems and because we have previously shown that Wld S protects dopaminergic terminal fields from MPTP in vivo, we used a dissociated midbrain culture system to determine the mechanism of Wld S -mediated neurite protection in dopamine neurons. Here, we show that, regardless of its enzymatic activity, the entire Wld S sequence is needed for the Wld S ' neuroprotective phenotype in dopaminergic neurons. Our data also illustrate that NAD+ has a neuroprotective effect that is different from Wld S -mediated protection.
Wld S protects cell bodies and neurites from MPP+
Cytoplasmic Wld S protects cell bodies and neurites against MPP+
Nmnat1 does not protect against MPP+toxicity
Inactive Wld S protects cell bodies and neurites against MPP+
NAD+ protects cell bodies and neurites against MPP+
Sirt1 is not responsible for the NAD-mediated protection of cell bodies and neurites against MPP+
NAD+ and Wld S effects are additive
The mechanism(s) by which Wld S protects axons is still unclear. Peripheral model studies underscore the role of Nmnat and its product, NAD+, in protecting axons from various injuries whereas few central nervous system studies have been done. Using cellular, molecular and pharmacological tools, the present findings show that the chimeric Wld S gene product plays a critical role in protecting dopaminergic processes, one not dependent upon Nmnat activity. Specifically, neither Nmnat, cytoplasmically-targeted Nmnat, nor Nmnat 3 were able to prevent toxicity associated with the dopaminergic toxin MPP+ whereas, akin to previous reports [27, 28], cyto Nmnat protected DRG axons from known axonal toxins. In contrast, Wld S , cytoplasmically-expressed Wld S , and Wld S with an inactive Nmnat domain, all significantly protected dopaminergic neurites from toxin-mediated loss. Despite the inability of Nmnat to protect dopamine processes, NAD+ and its precursor, Nmn, were neuroprotective. As Wld S and NAD+ were additive in this model system, current results suggest that these protectants act through separate, possibly parallel pathways. This is in agreement with previous findings by Wishart et. al. (2008)  showing that Wld S increases expression of cell cycle-related genes through both NAD+-dependent and independent pathways. Thus, NAD+ or its derivatives as well as Wld S and its targets protect dopamine processes and may aid in the development of therapeutics preserving the connections and circuitry important in PD.
The role of Nmnat and NAD+ in recapitulating the full effect of Wld S has been controversial. In vitro studies have shown that overexpression of Nmnat1 by itself protects axons from many mechanical, genetic or toxin-induced injuries [20, 41]. In contrast, transgenic animals expressing nuclear Nmnat1 did not replicate the effects of Wld S [42, 43] whereas cytoplasmically  or axonally targeted Nmnat1  were equally if not more effective. Thus, site of action plays a role in Nmnat1's effectiveness . These data together with findings showing that the first 16 N-terminal amino acids of the Wld S gene product are required for full Wld S protection , possibly by redistributing enough Wld S to cytoplasmic or axonal compartments, are consistent with the notion that both the N-terminal portion of Wld S and Nmnat1 are necessary for full axonal protection .
The importance of Nmnat catalytic activity is reflected in several mutational studies in which Nmnat's active sites have been disrupted and neuroprotection was lost [26, 27, 29, 43]. Moreover, NAD+ itself and/or some of its biosynthetic precursors, protect against axonal degeneration in peripheral model systems as well as in experimental autoimmune encephalomyelitis (EAE); [28, 44], ischemia [45, 46], Alzheimer's disease , and PD [48–50]. In at least one study however, addition of NAD+ was not effective . Moreover, Drosophila Nmnat (dNmnat) did not require enzymatic activity for axon protection against insults such as excitotoxicity, polyglutamine-induced dysfunction, or mechanical injury  leading to the suggestion that dNmnat may perform a chaperone-like function . Indeed, structural studies of various Nmnats have revealed characteristic similarities to known chaperones such as UspA and Hsp100 . Consistent with this notion, dNmnat was recently shown to function as a stress protein in response to heat shock, hypoxia, and the mitochondrial Complex I toxin, paraquat . However, in dopaminergic neurons, Nmnat1 does not seem to function as either an axonal protectant or a chaperone.
Studies have indicated that MPP+ can block electron transport by acting at the same site as the Complex I inhibitor, rotenone, leading to the production of free radical species and a loss of ATP production [53–55]. MPP+ affects other processes as well including the rapid release of dopamine from vesicular stores [56, 57]; depolymerization of microtubules [16, 58]; induction of autophagy [19, 59], and the rapid loss of mitochondrial membrane potential and reduction in mitochondrial motility in dopamine axons . Since many of these effects involve mitochondrial function, conceivably the Wld S gene product is involved in preserving mitochondrial health or maintaining homeostatic control. Recently, Barrientos et al. reported that Wld S is able to regulate the mitochondrial permeability transition pore (PTP) preventing, amongst other things, calcium release, ATP loss, oxidative stress and release of proteins involved in axonal degeneration . This is consistent with Wishart et al. (2007) showing that synaptosomes isolated from Wld S versus wild type animals expressed higher levels of various mitochondrial proteins including the PTP protein, VDAC2 . Barrientos et al. suggested that Wld S is part of a regulatory cascade that also involves JNK activation upstream of PTP opening . Although JNK is a known regulator of axon degeneration in DRGs , it has been reported to not play a role in 6-OHDA-mediated degeneration of the striatum . In addition, we have recently showed that the JNK inhibitor, SP600125, did not prevent MPP+ effects on dopaminergic mitochondria . Thus diverse, unknown, regulatory steps appear to mediate Wld S effects in dopamine axons.
Given its role as a ubiquitous cofactor, NAD+ influences many cellular decisions such as DNA damage repair  and transcriptional regulation and differentiation . Earlier studies suggested that increased NAD+ levels led to SIRT activation which, in turn, activated a transcription factor that induced genes involved in neuroprotection [27, 32]. Although an attractive hypothesis, subsequent studies using SIRT1 knock out animals did not support this notion for DRG neurons , or as in the present study, for dopaminergic neurons (Figure 8).
Unlike what we have reported in vivo , dopaminergic cell death from MPP+ treatment was also attenuated in Wld S cultures (Figure 1). Given that Wld S is known to protect axons and synapses from injury, it is possible that it can also indirectly protect cell bodies. Similar indirect effects on cell bodies have been reported by Gillingwater et. al. (2004) in both the caudate nucleus and hippocampus of Wld S mice following transient global ischemia . More recently, a cytoplasm-targeted Nmnat transgenic mouse protected cell bodies and processes from NMDA-mediated excitotoxicity . Authors of the latter study speculate that Nmnat can potentially influence a common pathway, albeit one not tied to caspase 3 activation, in certain neurons. Perhaps a similar pathway is activated in other systems as well since Wld S also protects motoneuron cell bodies in a mouse model of progressive motor neuropathy .
Why are results in dopamine neurons different than other systems? Because many of the studies published have been performed in peripheral model systems with dramatically over-expressed protein, there may be neuronal-specific or expression level-related effects that might account for the differences. For example, Wld S has shown protection in several central nervous system models, but few have been further tested with only Nmnat1 even in dissociated neuronal models . Then too, dopaminergic axons may have intrinsic differences that contribute to the Wld S effect. For instance, dopamine neurons have fewer , smaller and slower mitochondria than non-dopaminergic neurons . Moreover, dopamine neurons produce a neurotransmitter prone to oxidation , exhibit a greater dependence on L-type Ca2+ channels with subunits that result in deleterious amounts of intracellular calcium and ensuing mitochondrial dysfunction , and extend long, thin lightly-myelinated processes which are selectively vulnerable in PD . This suggests that dopaminergic neurons may be more vulnerable to insults that affect mitochondrial function. Given that enzymatically inactive Wld S is able to protect dopaminergic, but not DRG neurons, it is possible that Wld S also protects mitochondria in a manner independent of its NAD+-synthesizing ability. This as yet unknown function of Wld S may be unmasked in dopaminergic neurons due to their unique phenotype. In contrast, NAD+ may contribute more to Wld S -mediated protection in non-DA neurons.
In support of our previous in vivo study showing that Wld S protects dopaminergic terminal fields from MPTP, the current results demonstrate in dissociated dopamine cultures that the entire Wld S sequence is needed for axonal protection, regardless of its NAD+-synthesizing activity. Indeed, NAD+ and Wld S act through separate, possibly parallel, mechanisms to protect dopamine axons. As MPP+ is thought to impair mitochondrial function, in agreement with other studies, our results suggest that Wld S might be involved in preserving mitochondrial health or maintaining cellular metabolism. Given that Parkinson's disease is the second most common neurodegenerative disorder, our findings support the idea that studies expanding therapeutic efforts towards maintaining connections as well as saving the cell body will help in developing better interventions for PD.
Materials and methods
Cell culture and toxin treatment
For primary midbrain cultures, the ventral mesencephalon was removed from embryonic day 14 (E14) murine embryos as previously described [33, 72]. Wild-type (C57/Bl6) and Wld S (C57Bl/OlaHsd-WldS) mice were ordered from Harlan (Bichester, UK). Sirt1 knockout mice were obtained from Dr. Christian Sheline (Louisiana State University - Health Science Center, New Orleans, LA). Cyto Wld S mice were obtained from Dr. Michael Coleman (Babraham Institute, UK) . Animals were treated in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All procedures were approved by the Washington University School of Medicine animal experimentation committee. Plates were pre-coated overnight with 0.2 mg/ml poly-D-lysine (Sigma-Aldrich, St. Louis, MO). Cells were plated at a density of approximately 125,000 cells/cm2 and maintained in serum-free Neurobasal medium (Invitrogen, Carlsbad, CA) supplemented with 1× B27 supplement (Invitrogen), 0.5 mM L-glutamine (Sigma-Aldrich), and 0.01 μg/ml streptomycin plus 100 U penicillin. Half of the culture medium was replaced with fresh Neurobasal medium after 5 days in vitro (DIV). Cultures were pretreated with 1 mM NAD+, 1 mM NMN, 1 mM nicotinic acid mononucleotide (NaMN), or a comparable volume of vehicle 24 hours before toxin treatment. Cultures were treated with either 1 μM 1-methyl-4-phenylpyridinium (MPP+), the active metabolite of MPTP or vehicle on DIV 7. Dorsal root ganglion (DRG) cells were obtained from E14 murine embryos as previously described . Cells were plated on coverslips precoated with 0.1 mg/ml poly-L-ornithine (Invitrogen) and 32 μg/ml laminin-1 (Invitrogen) and maintained in DRG media which consisted of Eagle Minimal Essential Media (Invitrogen) supplemented with chick embryo extract (Invitrogen), 10% fetal calf serum (Invitrogen), 50 ng/ml Nerve Growth Factor (Harlan Biosciences, Madison, WI) and 50 U/ml penicillin-50 g/ml streptomycin. Half of the culture medium was replaced with fresh DRG medium after DIV 5. After transduction with lentivirus on DIV 2, DRG cultures were treated with 0.4 μM vincristine or vehicle on DIV 7. NAD+, NMN, NaMN, MPP+, and vincristine were all obtained from Sigma-Aldrich.
Lentiviral infection of dopaminergic neurons
The lentiviral expression plasmids FUGW, FCIV-WldS, FCIV-Nmnat1, FCIV-Ube4b, FCIV-Nmnat3, FCIV- Nmnat1(W170A), FCIV-cytNmnat1, and FCIV-WldS(W258A) were obtained from Dr. Jeffrey Milbrandt (Washington University, Saint Louis). Lentiviruses expressing transgenes were generated by the Hope Center for Neurological Disorders Viral Core (Washington University, Saint Louis). For infection of DRG and primary midbrain neurons, 50 μl lentivirus (105 infectious units/μl) was added to the well of a 7-mm dish containing approximately 70,000 neurons on DIV 2. Transduced primary midbrain and DRG neurons were treated with MPP+ and vincristine, respectively, on DIV 7. Viral infection and transgene expression was monitored using the GFP reporter via fluorescent microscopy.
Primary dopaminergic cultures and DRGs were plated in 7 mm microwell plates (MatTek Corp., Ashland, MA). Cells treated with MPP+ were fixed with 4% paraformaldehyde (PFA) in PBS after 48 hours. Cultures were stained with sheep polyclonal anti-tyrosine hydroxylase (TH) (Novus Biologicals, Littleton, CO) and Cy3 α-sheep (Molecular Probes, Carlsbad, CA). Localization of cytoplasmic Wld S was confirmed using rabbit Wld S antibody (gift of M.P. Coleman) and Alexa488 α-rabbit (Molecular Probes). TH+ cells and neurites were counted using unbiased stereological methods (Stereo Investigator, MicroBrightField, Williston, VT). DRG cultures treated with vincristine were subsequently stained with mouse acetylated tubulin (Sigma-Aldrich) and Cy3 α-mouse (Molecular Probes). Neurites were counted as described above. All images were acquired by confocal microscopy (Olympus Fluoview 500, Olympus, Center Valley, PA) and processed in ImageJ (NIH).
Primary midbrain cultures were plated in 48-well plates and transduced with the transgene of interest as described above. Lysates were collected in RIPA buffer (150 mM NaCl, 1% Nonidet P-40, 0.5% NaDoc, 0.1% SDS, 50 mM Tris pH 8.0) with protease inhibitor mixture (Roche, Mannheim, Germany) and incubated on ice for 30 minutes. Insoluble cell debris was removed by centrifugation and the protein concentration of each cell lysate was determined by Bradford protein assay (BioRad, Hercules, CA). Equal amounts of protein were run on SDS-polyacrylamide gels and transferred to polyvinylidene diflouride (PVDF) membranes (BioRad). PVDF membranes were probed with either rabbit Wld S antibody or chicken polyclonal anti-GFP antibody (Aves Labs, Tigard, OR). As a control, PVDF membranes were also probed with goat polyclonal anti-HRP60 antibody (Santa Cruz Biotechnology, Santa Cruz, CA). The secondary antibodies used were either a HRP-linked rabbit antibody or HRP-linked anti-chicken antibody and a HRP-linked anti-goat antibody (Jackson Immunoresearch, West Grove, PA). Membranes were developed with enhanced chemiluminescence (Amersham Biosciences), imaged with either a Storm PhosphorImager (Molecular Dynamics) or a ChemiDoc XRS System (Bio-Rad, Hercules, CA) and band intensities were determined using ImageQuant software (Amersham Biosciences).
Quantification of Cells and Neurites
TH+ cells and neurites were counted using unbiased stereological methods  (Stereo Investigator (MicroBrightfield, Williston, VT), in combination with a Zeiss Axioplan2 microscope (Thornwood, NY) and an Optronics Microfire camera. The number of counting sites necessary to achieve a coefficient of error < 0.1 was determined by preliminary experiments. The total number of TH+ cell bodies was calculated using the Fractionator function on Stereo Investigator by dividing the estimated number of cells by the estimated volume (μm3) of the dish sampled. Using the Petrimetrics function on Stereo Investigator, TH+ neurites intersecting the boundary of the Petrimetric probe were counted. Neurite length was derived by dividing the total estimated neurite length (μm) by the estimated volume (μm3) of the dish sampled
GraphPad Prism software (San Diego, CA) was used for statistical analysis. All data was collected from a minimum of three independent experiments done in triplicate. The significance of effects between control and experimental conditions was determined by a Student t-test or one-way ANOVA with Bonferroni Multiple Comparisons tests.
This work was supported by National Institutes of Health Grants NS39084 (K.L.O.) and National Institutes of Health Neuroscience Blueprint Core Grant NS057105 to Washington University. This work was also supported by the Bakewell Family Foundation. We thank Steven K. Harmon for technical support and Drs. Michael Coleman, Jeffrey Milbrandt, Christian Sheline, Valeria Cavalli and Jeong Sook Kim-Han for materials and helpful discussions.
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