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T-Cell-Mediated Disruption of the Neuronal Microtubule Network

2006; Elsevier BV; Volume: 169; Issue: 3 Linguagem: Inglês

10.2353/ajpath.2006.050791

1525-2191

Leah P. Shriver, Bonnie N. Dittel,

Microtubule and mitosis dynamics

During the course of the central nervous system autoimmune disease multiple sclerosis (MS), damage to myelin leads to neurological deficits attributable to demyelination and conduction failure. However, accumulating evidence has indicated that axonal injury is also a predictor of MS clinical disease. Using the animal model of MS, experimental autoimmune encephalomyelitis (EAE), we examined whether axonal dysfunction occurred early in disease and correlated with disease symptoms. We tracked axons during EAE by using transgenic mice that express yellow fluorescent protein (YFP) in neurons. At the onset of disease, we observed a loss of YFP fluorescence in the spinal cord in areas that coincided with immune cell infiltration, before prominent demyelination. These inflammatory lesions also exhibited evidence of axonal injury but not axonal loss. During the recovery phase of EAE, the return of YFP fluorescence occurred in parallel with the resolution of inflammation. Using in vitro cultured neurons expressing YFP, we demonstrated that encephalitogenic T cells alone directed the destabilization of microtubules within neurites, resulting in a change in the pattern of YFP fluorescence. This study provides evidence that encephalitogenic T cells directly cause reversible axonal dysfunction at the onset of neurological deficits during an acute central nervous system inflammatory attack. During the course of the central nervous system autoimmune disease multiple sclerosis (MS), damage to myelin leads to neurological deficits attributable to demyelination and conduction failure. However, accumulating evidence has indicated that axonal injury is also a predictor of MS clinical disease. Using the animal model of MS, experimental autoimmune encephalomyelitis (EAE), we examined whether axonal dysfunction occurred early in disease and correlated with disease symptoms. We tracked axons during EAE by using transgenic mice that express yellow fluorescent protein (YFP) in neurons. At the onset of disease, we observed a loss of YFP fluorescence in the spinal cord in areas that coincided with immune cell infiltration, before prominent demyelination. These inflammatory lesions also exhibited evidence of axonal injury but not axonal loss. During the recovery phase of EAE, the return of YFP fluorescence occurred in parallel with the resolution of inflammation. Using in vitro cultured neurons expressing YFP, we demonstrated that encephalitogenic T cells alone directed the destabilization of microtubules within neurites, resulting in a change in the pattern of YFP fluorescence. This study provides evidence that encephalitogenic T cells directly cause reversible axonal dysfunction at the onset of neurological deficits during an acute central nervous system inflammatory attack. Multiple sclerosis (MS) is an autoimmune inflammatory disease of the central nervous system (CNS) of unknown etiology. It is the leading cause of neurological disability in young adults and affects more than 1.1 million individuals worldwide.1Lassmann H Axonal injury in multiple sclerosis.J Neurol Neurosurg Psychiatry. 2003; 74: 695-697Crossref PubMed Scopus (94) Google Scholar, 2Noseworthy JH Lucchinetti C Rodriguez M Weinshenker BG Multiple sclerosis.N Engl J Med. 2000; 343: 938-952Crossref PubMed Scopus (3027) Google Scholar Lesions in the CNS of MS patients are characterized by an inflammatory infiltrate consisting primarily of T cells and macrophages surrounding demyelinated plaques.3Kornek B Storch MK Weissert R Wallstroem E Stefferl A Olsson T Linington C Schmidbauer M Lassmann H Multiple sclerosis and chronic autoimmune encephalomyelitis: a comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions.Am J Pathol. 2000; 157: 267-276Abstract Full Text Full Text PDF PubMed Scopus (789) Google Scholar Loss of conduction along demyelinated nerve fibers is believed to play a role in the pathogenesis of MS; however, recent studies have renewed interest in axonal pathology as a mediator of clinical symptoms.4Medana IM Esiri MM Axonal damage: a key predictor of outcome in human CNS diseases.Brain. 2003; 126: 515-530Crossref PubMed Scopus (364) Google Scholar, 5Bjartmar C Wujek JR Trapp BD Axonal loss in the pathology of MS: consequences for understanding the progressive phase of the disease.J Neurol Sci. 2003; 206: 165-171Abstract Full Text Full Text PDF PubMed Scopus (491) Google Scholar In brain biopsies taken from newly diagnosed MS patients, axonal injury, detected by the presence of amyloid precursor protein (APP) aggregates, was observed in the early stages of disease in both active demyelinating plaques as well as in normal-appearing white matter.3Kornek B Storch MK Weissert R Wallstroem E Stefferl A Olsson T Linington C Schmidbauer M Lassmann H Multiple sclerosis and chronic autoimmune encephalomyelitis: a comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions.Am J Pathol. 2000; 157: 267-276Abstract Full Text Full Text PDF PubMed Scopus (789) Google Scholar, 6Kuhlmann T Lingfeld G Bitsch A Schuchardt J Brück W Acute axonal damage in multiple sclerosis is most extensive in early disease stages and decreases over time.Brain. 2002; 125: 2202-2212Crossref PubMed Scopus (658) Google Scholar These studies suggested that the myelination status of the neuron is not the sole predictor of vulnerability to injury in diseases such as MS. The relationship between inflammation, oligodendrocyte damage, and neuronal dysfunction is complex, and the mechanisms that lead to tissue damage and clinical symptoms are not well understood. Furthermore, recent evidence from magnetic resonance imaging studies has shown that current treatments such as β-interferon do not stop the accumulation of axonal injury throughout time.7Parry A Corkill R Blamire AM Palace J Narayanan S Arnold D Styles P Matthews PM Beta-interferon treatment does not always slow the progression of axonal injury in multiple sclerosis.J Neurol. 2003; 250: 171-178Crossref PubMed Scopus (82) Google Scholar This study and others suggest that current therapies may be inadequate at treating the degenerative component of MS. Experimental autoimmune encephalomyelitis (EAE), the animal model for MS, has been used to study the mechanisms of axonal damage during CNS inflammation. In this model, immunization of mice with myelin proteins in adjuvant or the transfer of activated myelin-specific T cells induces an ascending paralytic disease that mimics some MS symptoms. CNS lesions in EAE are also composed of inflammatory cells accompanied by demyelination that primarily occurs in the white matter tracts of the spinal cord. During EAE, axonal damage has been observed in both early and late active demyelinating plaques.3Kornek B Storch MK Weissert R Wallstroem E Stefferl A Olsson T Linington C Schmidbauer M Lassmann H Multiple sclerosis and chronic autoimmune encephalomyelitis: a comparative quantitative study of axonal injury in active, inactive, and remyelinated lesions.Am J Pathol. 2000; 157: 267-276Abstract Full Text Full Text PDF PubMed Scopus (789) Google Scholar However, in the Theiler's murine encephalomyelitis virus model of viral-induced demyelination, damaged axons, detected by an increase in nonphosphorylated neurofilament-H, were present before the onset of demyelination.8Tsunoda I Kuang LQ Libbey JE Fujinami RS Axonal injury heralds virus-induced demyelination.Am J Pathol. 2003; 162: 1259-1269Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar Therefore, it is unclear the extent to which neuronal dysfunction precedes myelin degeneration in EAE and MS. The ability to track axonal degeneration or functional loss during disease has been difficult because of lack of reagents that label both neuronal cell bodies and processes. Fluorescent tracers are useful, but consistent labeling is technically difficult in mice. Previous studies have demonstrated that axonal injury can be visualized using two antibodies, APP9Umehara F Abe M Koreeda Y Izumo S Osame M Axonal damage revealed by accumulation of β-amyloid precursor protein in HTLV-I-associated myelopathy.J Neurol Sci. 2000; 176: 95-101Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar and SMI-32.10Trapp BD Peterson J Ransohoff RM Rudick R Mork S Bö L Axonal transection in the lesions of multiple sclerosis.N Engl J Med. 1998; 338: 278-285Crossref PubMed Scopus (3485) Google Scholar These antibodies detect two different parameters of degeneration. SMI-32, which detects nonphosphorylated neurofilament-H, has previously been correlated with transected neurites or axonal swelling in MS lesions,10Trapp BD Peterson J Ransohoff RM Rudick R Mork S Bö L Axonal transection in the lesions of multiple sclerosis.N Engl J Med. 1998; 338: 278-285Crossref PubMed Scopus (3485) Google Scholar whereas APP-positive axons are believed to occur when axonal transport is disrupted leading to an accumulation of APP that reacts with the antibody.11Mori I Goshima F Mizuno T Imai Y Kohsaka S Ito H Koide N Yoshida T Yokochi T Kimura Y Nishiyama Y Axonal injury in experimental herpes simplex encephalitis.Brain Res. 2005; 1057: 186-190Crossref PubMed Scopus (8) Google Scholar, 12Coleman MP Adalbert R Beirowski B Neuroprotective strategies in MS: lessons from C57BL/WldS mice.J Neurol Sci. 2005; 233: 133-138Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar Both SMI-32 and APP have previously been shown to correlate with axonal injury, neuritic swelling, and axonal transport defects during disease conditions in the CNS such as viral infections, Alzheimer's disease, and traumatic brain injury.4Medana IM Esiri MM Axonal damage: a key predictor of outcome in human CNS diseases.Brain. 2003; 126: 515-530Crossref PubMed Scopus (364) Google Scholar, 13Petzold A Neurofilament phosphoforms: surrogate markers for axonal injury, degeneration and loss.J Neurol Sci. 2005; 233: 183-198Abstract Full Text Full Text PDF PubMed Scopus (485) Google Scholar The integrity of the neuronal transmission is dependent on proper delivery to the axon terminal of synaptic vesicles and proteins that are synthesized in the cell body. In addition, viability is maintained by retrograde transport of neurotrophic factors from the synapse to the cell body.14Guzik BW Goldstein LS Microtubule-dependent transport in neurons: steps towards an understanding of regulation, function and dysfunction.Curr Opin Cell Biol. 2004; 16: 443-450Crossref PubMed Scopus (106) Google Scholar Because the axon terminal can be a significant distance from the cell body, delivery of critical factors occurs by the procession of molecular motors carrying cargo on a network of microtubules. Microtubules consist of α- and β-tubulin that form a tubular protofilament. Motor proteins such as dynein and kinesin associate with these filaments and deliver vesicles and proteins in both the anterograde and retrograde direction.15Hirokawa N Takemura R Molecular motors in neuronal development, intracellular transport and diseases.Curr Opin Neurobiol. 2004; 14: 564-573Crossref PubMed Scopus (123) Google Scholar, 16Hirokawa N Takemura R Molecular motors and mechanisms of directional transport in neurons.Nat Rev Neurosci. 2005; 6: 201-214Crossref PubMed Scopus (677) Google Scholar The stability of microtubules in the neuritic processes is maintained by microtubule-associated proteins. The disruption of microtubule integrity that has been observed in Huntington's chorea and the early stages of Wallerian degeneration was associated with axonal transport defects resulting in the accumulation of cytosolic proteins in discreet domains along the axon, termed a "beads on a string" pattern.12Coleman MP Adalbert R Beirowski B Neuroprotective strategies in MS: lessons from C57BL/WldS mice.J Neurol Sci. 2005; 233: 133-138Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar, 17Trushina E Dyer RB Badger II, JD Ure D Eide L Tran DD Vrieze BT Legendre-Guillemin V McPherson PS Mandavilli BS Van Houten B Zeitlin S McNiven M Aebersold R Hayden M Parisi JE Seeberg E Dragatsis I Doyle K Bender A Chacko C McMurray CT Mutant huntingtin impairs axonal trafficking in mammalian neurons in vivo and in vitro.Mol Cell Biol. 2004; 24: 8195-8209Crossref PubMed Scopus (417) Google Scholar Thus, dysregulation of microtubule stability is an early event during neurodegeneration. In MS, neither the temporal relationship nor the interrelatedness of CNS pathologies—including immune cell infiltration, neuronal damage/dysfunction, and demyelination—are well understood or characterized. The understanding of how these pathological events are connected is important for the development of therapeutic modalities for the treatment of MS. One difficulty in tracking CNS events is the lack of markers for neuronal damage or dysfunction. The expression of green fluorescent protein (GFP) or its derivatives has revolutionized the tracking of cells and molecules. With the use of cell-specific promoters, GFP labeling can be used to delineate specific cell types during various experimental conditions. Therefore, to track neuronal health during EAE, we used the Thy1-yellow fluorescent protein (YFP) transgenic mouse, which expresses YFP in the cell body and dendritic and axonal processes in both motor and sensory neurons, providing a vital marker for neurons under various experimental conditions.18Feng G Mellor RH Bernstein M Keller-Peck C Nguyen QT Wallace M Nerbonne JM Lichtman JW Sanes JR Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.Neuron. 2000; 28: 41-51Abstract Full Text Full Text PDF PubMed Scopus (2427) Google Scholar Using an acute model of EAE associated with spontaneous recovery, we found that YFP fluorescence was lost from the white matter regions of the spinal cord at the onset of clinical symptoms, before evidence of demyelination. Areas of the spinal cord that lacked YFP fluorescence also contained inflammatory infiltrates. As EAE clinical disease progressed, demyelination was observed along with an increased loss of YFP fluorescence and the presence of damaged axons. Axonal damage was indicated by the detection of nonphosphorylated neurofilament-H and the accumulation of APP. On recovery from EAE clinical disease, the inflammatory lesions resolved and were accompanied by remyelination and the return of YFP fluorescence. Using electron microscopy (EM), we found that there was no overt loss of axons during the acute disease course. Furthermore, we showed that encephalitogenic T cells disrupt microtubule integrity in cultured neurons. Therefore, the loss of YFP fluorescence in inflammatory lesions is a good indicator of EAE CNS pathology and demonstrates that functional changes occur in neurons during inflammation in the CNS, which are likely mediated by T cells. Thus, neuronal dysfunction likely contributes to many of the clinical symptoms in MS and EAE, and identifying early events in neuronal degeneration may help to develop therapies that delay the progression of disease and promote remission of clinical symptoms. B10.PL (H-2u), B6.Cg-Tg(Thy1-YFP)16Jrs/J (Thy1-YFP), FVB, and Tg(TcrHEL3A9)Mmd/J (HEL-TCR) mice were purchased from the Jackson Laboratory (Bar Harbor, ME).18Feng G Mellor RH Bernstein M Keller-Peck C Nguyen QT Wallace M Nerbonne JM Lichtman JW Sanes JR Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP.Neuron. 2000; 28: 41-51Abstract Full Text Full Text PDF PubMed Scopus (2427) Google Scholar The Thy1-YFP founder mice were on the C57BL/6 background and were backcrossed to B10.PL for five generations. (Thy1-YFPxFVB)F1 mice were generated in our colony. The myelin basic protein (MBP)-T-cell receptor (TCR) transgenic mice expressing a TCR transgene specific for the acetylated NH2-terminal peptide of MBP (Ac1-11) were generated as previously described.19Dittel BN Merchant RM Janeway Jr, CA Evidence for Fas-dependent and Fas-independent mechanisms in the pathogenesis of experimental autoimmune encephalomyelitis.J Immunol. 1999; 162: 6392-6400PubMed Google Scholar The Ac1-11 (Ac-ASQKRPSQRSK) and HEL 46-61 (NTDGSTDYGILQINSR) peptides were synthesized and high performance liquid chromatography purified by the Peptide Core Laboratory at BloodCenter of Wisconsin, Blood Research Institute. The anti-mouse antibodies for CD11b, the β chain of the TCR, IgG1, and IgG2b were purchased from eBioscience (San Diego, CA). The SMI-99 and SMI-32 monoclonal antibodies that detect MBP and a nonphosphorylated epitope of neurofilament-H, respectively, were purchased from Sternberger Monoclonals (Lutherville, MD). Anti-β tubulin was purchased from Sigma-Aldrich (St. Louis, MO) or Chemicon (Temecula, CA). Total splenocytes from MBP-TCR (I-Au restricted) and HEL-TCR (I-Ak restricted) transgenic mice were activated, as described,19Dittel BN Merchant RM Janeway Jr, CA Evidence for Fas-dependent and Fas-independent mechanisms in the pathogenesis of experimental autoimmune encephalomyelitis.J Immunol. 1999; 162: 6392-6400PubMed Google Scholar in the presence of 5 μg/ml Ac1-11 or the HEL 46-61, respectively. Naïve splenocytes were isolated from B10.PL mice and, for some experiments, were cultured overnight in 2.5 μg/ml concanavalin A. EAE was induced by the adoptive transfer of MBP-specific encephalitogenic T cells generated as described.19Dittel BN Merchant RM Janeway Jr, CA Evidence for Fas-dependent and Fas-independent mechanisms in the pathogenesis of experimental autoimmune encephalomyelitis.J Immunol. 1999; 162: 6392-6400PubMed Google Scholar Briefly, 1 × 106 activated MBP-TCR T cells were intravenously injected into irradiated (360 rads) 5- to 8-week-old B10.PL or Thy1-YFP recipients. Animals were assessed daily for clinical symptoms and scored using a scale from 1 to 5 as follows: 0, no disease; 1, limp tail and/or hind limb ataxia; 2, hind limb paresis; 3, hind limb paralysis; 4, hind and forelimb paralysis; and 5, death. On days 10, 15, and 30 after induction of EAE, three mice were deeply anesthetized and perfused intracardially with 0.1 mol/L phosphate buffer (PB), followed by a paraformaldehyde-lysine-periodate fixative. Brains and spinal cords were harvested and fixed overnight at 4°C in paraformaldehyde-lysine-periodate fixative. After fixation, the tissue was washed in 0.1 mol/L PB and cryoprotected in solutions of 10 and 20% sucrose. The tissue was snap-frozen at −80°C in Tissue-Tek OCT (Sakura, Torrance, CA). Transverse sections of spinal cord at 10-μm thickness were used for immunofluorescence. Images of lumbar spinal cord were taken using Meta Morph software (Universal Imaging Corp., Downingtown, PA) from three individual mice at each time point (untreated, and days 10, 15, 22, and 35). The average fluorescence intensity in the columns was measured and expressed as mean ± SD. Statistical significance was determined using a one-way analysis of variance for multiple comparisons. Significance thresholds were P < 0.05. Sections of spinal cord were rehydrated in 0.1 mol/L PB with 0.01% Triton for 10 minutes. If a biotinylated primary antibody was used, sections were blocked with an avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA) followed by a 1-hour incubation in 100% fetal calf serum. The primary antibodies anti-CD11b-biotin (1:75), anti-TCRβ-phycoerythrin (1:75), and SMI-99 (1:100) were diluted in 30% fetal calf serum/0.1 mol/L PB and incubated for 2 hours. After washing in 0.1 mol/L PB, sections with anti-CD11b-biotin were incubated with streptavidin-Alexa Fluor 350 (1:250) (Molecular Probes, Eugene, OR) for 1 hour. After incubation with SMI-99, the slides were washed in 0.1 mol/L PB and rat anti-mouse IgG2b-Texas Red (1:200) was applied to sections for 1 hour. The slides were washed in 0.1 mol/L PB followed by a wash in distilled water and then coverslipped with Aquamount (Biomeda, Foster City, CA) and imaged using a Zeiss Axiostop microscope and Sensys camera with Meta Morph imaging software (Universal Imaging Corp.). Images represent spinal cords taken from three separate experiments. For neuronal cultures, 4% paraformaldehyde for 10 minutes was used for fixation and treated as for frozen sections, staining with anti-β-tubulin for 2 hours followed by a 1-hour incubation with anti-mouse IgG1-Texas Red. Control, irradiation-only mice, and mice with EAE on days 10, 15, and 30 were deeply anesthetized and then perfused intracardially with PB followed by a solution of ice-cold 2.5% glutaraldehyde in 0.1 mol/L cacodylate buffer. Spinal cords were dissected and fixed overnight in 2.5% glutaraldehyde. Ultrathin sections were prepared and toluidine blue staining and EM were performed by the EM facility at the Medical College of Wisconsin (Milwaukee, WI). Three mice per group were analyzed at each time point. Embryonic neurons were cultured using an adapted protocol from Ransom and colleagues.20Ransom BR Neale E Henkart M Bullock PN Nelson PG Mouse spinal cord in cell culture. I. Morphology and intrinsic neuronal electrophysiologic properties.J Neurophysiol. 1977; 40: 1132-1150Crossref PubMed Scopus (419) Google Scholar Brains and spinal cords were isolated on embryonic day 15 from Thy1-YFP or (Thy1-YFPxFVB)F1 mice and titrated in DISGH buffer (135 mmol/L NaCl, 5 mmol/L KCL, 0.3 mmol/L Na2HPO4, 0.2 mmol/L KH2PO4, 16.5 mmol/L glucose, 22 mmol/L sucrose, and 9.86 mmol/L HEPES), followed by digestion in 0.67 mg/ml of papain at 37°C for 30 minutes. The tissue was then titrated in 40 μg of DNase diluted in minimal essential medium (Mediatech, Herndon, VA), and the cells were pelleted by centrifugation and resuspended in minimal essential medium containing 10% horse serum and 10% fetal calf serum. Neurons were plated on polyethylenimine-coated plates, and after 8 hours the medium was replaced with neurobasal media containing N2 supplement (Invitrogen, Carlsbad, CA), 2 mmol/L glutamine, 100 μg/ml gentamicin, and 2.5 μg of fungizone. Three days later, the cultures were treated with 0.054 mmol/L fluorodeoxyuridine (Sigma-Aldrich) and 0.014 mmol/L uridine (Sigma-Aldrich) to inhibit proliferation of astrocytes. Fresh medium was added to the culture every 2 days, and cultures were used ∼10 days after seeding. T cells and splenocytes were diluted in neurobasal medium before addition to the neuronal cultures. Live cell imaging was conducted using a Nikon fluorescent microscope with a Spot camera and Meta Morph Imaging Software (Universal Imaging Corp.). In experiments using taxol (paclitaxol, Sigma-Aldrich), neurons were pretreated for 1 hour with 10 μmol/L taxol and washed in neurobasal media, and then encephalitogenic T cells were added to the cultures. Colchicine (Sigma-Aldrich) (200 μmol/L) was added to Thy1-YFP cultures for 1 hour before live cell imaging. For some experiments, neurons were labeled with 10 μmol/L carboxy SNARF-1 acetate (Molecular Probes) for 30 minutes at 37°C and washed twice before addition of T cells. Because we backcrossed the Thy1-YFP mice onto the B10.PL background, we first determined the nature of the EAE disease course in the Thy1-YFP mice. As we previously reported for wild-type B10.PL mice,19Dittel BN Merchant RM Janeway Jr, CA Evidence for Fas-dependent and Fas-independent mechanisms in the pathogenesis of experimental autoimmune encephalomyelitis.J Immunol. 1999; 162: 6392-6400PubMed Google Scholar, 21Ponomarev ED Dittel BN γδ T cells regulate the extent and duration of inflammation in the central nervous system by a Fas ligand-dependent mechanism.J Immunol. 2005; 174: 4678-4687PubMed Google Scholar the Thy1-YFP mice exhibited a monophasic acute disease course (Figure 1A). The mice first showed signs of clinical disease 7 to 10 days after the adoptive transfer of the MBP-specific T cells, presenting with a weak tail or hind limb ataxia. The severity of disease increased until the peak of disease on days 13 to 16, at which time the mice exhibited hind limb paresis or paralysis. The mice then underwent spontaneous recovery completely resolving clinical symptoms by day 35 (Figure 1A). To determine the distribution of YFP fluorescence in the Thy1-YFP B10.PL mice, we examined spinal cords from untreated mice. Continuous YFP fluorescence was evident in the ventral, lateral, and dorsal white matter columns as well as in cell bodies of neurons in both dorsal and ventral horns (Figure 1B and data not shown). We next determined whether changes in YFP fluorescence could be detected in areas of the lumbar spinal cord where we have previously shown inflammatory lesions to reside in EAE in B10.PL mice.22Ponomarev ED Shriver LP Maresz K Dittel BN Microglial cell activation and proliferation precedes the onset of CNS autoimmunity.J Neurosci Res. 2005; 81: 374-389Crossref PubMed Scopus (330) Google Scholar At the onset of EAE clinical symptoms (day 10), small areas exhibiting loss of YFP fluorescence were observable in the white matter (Figure 1C). As EAE clinical disease progressed, the loss of YFP fluorescence became more prominent and was spread throughout the ventral, dorsal, and lateral columns by the peak of disease (Figure 1D). The YFP fluorescence that remained, surrounding areas of loss, exhibited a distinct punctate pattern (Figure 1D). Loss of YFP in these areas correlated well with the symptoms of sensory and motor loss in the lower extremities displayed by the mice. YFP fluorescence returned once the mice began to recover (Figure 1E); however, the punctate fluorescence pattern did not resolve until the mice showed signs of full recovery (Figure 1F). Changes in YFP fluorescence were quantified, and total YFP fluorescence decreased steadily until the peak of disease at which time the loss was statistically significant (Figure 1G). As observed in the histological sections, YFP fluorescence returned once the mice showed signs of clinical disease recovery (Figure 1G). These data indicate that the status of YFP fluorescence in neurons correlates with EAE clinical disease. To determine the relationship between the distribution of YFP fluorescence in the Thy1-YFP B10.PL mice and the presence of inflammatory cells, we examined spinal cords from untreated and mice at days 10, 15, 22, and 30 during EAE for the presence of T cells and macrophages. In control mice without EAE, continuous YFP fluorescence was observed (Figure 2A), whereas T cells bearing the TCRβ chain were absent (Figure 2F), and the CNS resident microglial cells exhibited dull CD11b staining (Figure 2, K and U). At both the onset and peak of disease, areas exhibiting loss of YFP fluorescence (Figure 2, B and C) also contained T cells (Figure 2, G and H) and macrophages (Figure 2, L and M). By overlaying YFP fluorescence with T cell and macrophage staining (Figure 2, Q and R), it is evident that loss of YFP fluorescence occurs only in the areas containing inflammatory infiltrates. This is particularly evident at high magnification, where an overlay of YFP fluorescence with CD11b staining at both the onset (Figure 2V) and peak of disease (Figure 2W) shows that areas with a complete loss of YFP fluorescence co-localized with macrophage accumulation. As the mice entered the recovery phase and were fully recovered, the inflammatory infiltrates resolved (Figure 2, I, J, N, O, S, and T) and YFP fluorescence returned (Figure 2, D and E). These data show that the loss of YFP fluorescence in axons can be used as an additional biological marker of EAE clinical disease and that it correlates with the presence of inflammatory infiltrates. Because the loss of YFP correlated with clinical disease and the presence of inflammatory infiltrates, we next determined whether YFP loss occurred in parallel with demyelination. In Thy1-YFP mice with no EAE, YFP fluorescence (Figure 3A) and the presence of myelin, as detected by staining for MBP (Figure 3F), were co-localized throughout the lumbar spinal cord (Figure 3K). On the day of EAE onset (day 10), when loss of YFP fluorescence was evident (Figure 3B), we were unable to detect signs of demyelination by staining for MBP (Figure 3G). Intact myelin was present in the lesions where loss of YFP was evident, as shown by a lack of yellow fluorescence in the overlay of YFP and MBP fluorescence (Figure 3L). A high-power magnification image, shown in Figure 3P, clearly demonstrates the presence of myelin in the absence of YFP fluorescence. These data suggest that loss of YFP fluorescence and demyelination are not temporally related. During peak of disease, areas of diminished YFP fluorescence were more prominent (Figure 3C), and these same regions exhibited loss of myelin (Figure 3H and overlay Figure 3M). Figure 3Q is a high-power image of the lesion at the ventral horn of Figure M showing that neurons that exhibit a punctate YFP fluorescence pattern are both myelinated (large arrow) and demyelinated (small arrow). As the clinical symptoms subsided in the recovery phase, a return of both YFP fluorescence in the axons along with the return of myelin was evident (Figure 3, D, I, and N). In recovered mice, both YFP fluorescence (Figure 3E) and the presence of MBP (Figure 3J) completely co-localized (Figure 3O). These data show that loss of YFP fluorescence is an earlier marker of clinical disease than demyelination, and that the two pathologies are likely independent events. Because the loss of YFP appeared before the detection of demyelination, we examined whether these areas also showed early evidence of neuronal dysfunction. Two antibodies, SMI-32 and anti-APP, have previously been shown to correlate with axonal damage.4Medana IM Esiri MM Axonal damage: a key predictor of outcome in human CNS diseases.Brain. 2003; 126: 515-530Crossref PubMed Scopus (364) Google Scholar Antibodies specific for APP detect early axonal dysfunction in MS lesions,6Kuhlmann T Lingfeld G Bitsch A Schuchardt J Brück W Acute axonal damag