Synaptophysin is an abundant membrane protein of synaptic vesicles. The objective of this study was to determine the utility of identifying synaptophysin accumulations (spheroids/ovoids/bulbs) in CNS white matter as an immunohistochemical marker of axonal damage in demyelinating and neuroinflammatory conditions. We studied the cuprizone toxicity and Theiler’s murine encephalomyelitis virus (TMEV) infection models of demyelination and analyzed CNS tissue from patients with multiple sclerosis (MS). Synaptophysin colocalized with the amyloid precursor protein (APP), a well-known marker of axonal damage. In the cuprizone model, numerous pathological synaptophysin/APP-positive spheroids/ovoids were identified in the corpus callosum at the onset of demyelination; the extent of synaptophysin/APP-positive vesicle aggregates correlated with identified reactive microglia; during late and chronic demyelination, the majority of synaptophysin/APP-positive spheroids/ovoids resolved but a few remained, indicating persistent axonal damage; in the remyelination phase, scattered large synaptophysin/APP-positive bulbs persisted. In the TMEV model, only a few large- to medium-sized synaptophysin/APP-positive bulbs were found in demyelinated areas. In MS patient tissue samples, the bulbs appeared exclusively at the inflammatory edges of lesions. In conclusion, our data suggest that synaptophysin as a reliable marker of axonal damage in the CNS in inflammatory/demyelinating conditions.

INTRODUCTION

Neurons are highly specialized and polarized CNS cells with discrete cell bodies, numerous dendrites, and normally a single axon that can reach to a meter in length in humans; they can be several meters long in other mammals such as whales. Axons serve not only as “electrical mains” but also as “highway networks” to move different cargos such as mitochondria, synaptic neurotransmitters, and various peptides throughout the CNS. Proteins synthesized in the neuronal cell body (anterograde transport) or absorbed via endocytosis at distal sites (retrograde transport) are delivered to their target places on microtubule “roads” via motor proteins such as kinesin (1, 2) or dynein (3, 4) by energy-dependent axonal transport (5, 6). Fast axonal transport (100–400 mm/day, 1–5 µm/s) mainly moves vesicular cargos whereas slow axonal transport (0.2–5 mm/day, 0.0002–0.05 µm/s) conveys mostly cytosolic and cytoskeletal proteins, such as microtubules, neurofilaments, and actin (5–8).

Axonal transport impairment and/or axonal damage are prominent features of CNS neuroinflammatory and neurodegenerative diseases, such as multiple sclerosis (MS) (9, 10). In neuropathological investigations of MS and CNS trauma, axonal damage is most often identified using immunohistochemical staining for the β-amyloid precursor protein (APP), a ubiquitously expressed 110–135 kDa integral glycoprotein type 1 with an extracellular N-terminus (11–13). The precise biological function(s) of APP is(are) still not well defined but different biological roles including regulation of growth pattern, differentiation, cell adhesion, and apoptosis in different cell types have been proposed. Synaptotrophic and neuroprotective functions have also been postulated (14, 15). APP undergoes kinesin-dependent, predominantly anterograde fast axonal transport (16, 17). Axonal damage/transection results in disturbances of this transport leading to accumulation of vesicles and subsequent formation of APP-positive spheroids or bulbs (18, 19).

The antibody SMI-32, which detects non-phosphory-lated neurofilaments, is another established marker for axonal damage (20–23). Neurofilaments are major components of the neuronal cytoskeleton; their phosphorylation is tightly linked to axonal transport and myelination (24–27). Neurofilaments are moved via slow axonal transport (8, 28, 29). Because of aberrant phosphorylation or impaired transport, neurofilaments also accumulate in affected axons in various pathological conditions (21, 30–32). The pathological mechanisms of axonal damage are not completely understood and additional markers to detect axonal disturbances are needed.

Synaptophysin is a 38-kDa integral transmembrane protein with 4 transmembrane domains (cytosolic C and N termini) and a major small synaptic vesicle protein comprising 7%–10% of the total synaptic vesicle proteins (33–35). It is a major Ca2+- and cholesterol-binding protein of synaptic vesicles (36–38), and it interacts with different synaptic vesicle and motor proteins including v-SNARE vesicle-associated, membrane protein 2/synaptobrevin II (VAMP2), vesicular proton pump V-ATPase, myosin V, dynamin I, and adaptor protein 1 (AP-1) (39–44). Synaptophysin is involved in different steps of synaptic biogenesis, vesicle protein sorting, vesicle priming, synapse formation, and exo- and endocytosis (37, 38, 45–48). It is transported in the fast axonal component in both anterograde and retrograde directions (6, 49, 50).

In this study, we investigated the potential for synaptophysin to serve as a marker for CNS axonal damage using 2 animal models that mimic different aspects of the complex processes in MS and in brain tissue from patients with MS.

Materials and Methods

Animals

Mice underwent routine cage maintenance once a week and were microbiologically monitored according to the Federation of European Laboratory Animal Science Associations recommendations (51). All research procedures were approved by the Review Board for the Care of Animal Subjects of the district government (Lower Saxony, Germany) and performed according to international guidelines on the use of laboratory animals. Male C57BL/6 mice were purchased from Charles River Laboratories (Sulzfeld, Germany); female SJL/JHanHsd mice were purchased from Harlan (Rossdorf, Germany). Control animals did not display any abnormalities during experiments.

Induction of Demyelination via Cuprizone and Tissue Processing

Experimental toxic demyelination was induced by feeding 8-week-old male C57BL/6 mice with 0.2% (w/w) cuprizone (bis-cyclohexanone oxaldihydrazone, Sigma-Aldrich, St. Louis, MO) mixed into a ground standard rodent chow (maintenance diet, rats/mice, Altromin, Lage, Germany) (52). The drinking water was prepared in the animal facility of the Hannover Medical School by demineralizing, filtering (5 µm) and bottling with a bottle filling system (Scanbur BK, Denmark). Cuprizone treatment was maintained for 5 weeks to investigate acute demyelination (53). Chronic demyelination was induced by feeding cuprizone for 12 weeks (54). After withdrawal of cuprizone, remyelination was analyzed 2 and 3 weeks later (ie week 7 and 8 after onset of experiments). After the 12-week period of cuprizone feeding, remyelination was investigated 2 and 4 weeks after withdrawal of the toxin (week 14 and 16 after onset of experiments). Tissue processing was performed as previously described (55, 56). At different time points, the mice under deep anesthesia were perfused transcardially with 4% paraformaldehyde ([PFA], Merck) for immunohistochemistry or with phosphate-buffered saline for real-time polymerase chain reaction (PCR) analyses.

For immunohistochemistry, brains were post-fixed in 4% PFA and paraffin-embedded. Seven-μm serial coronal sections were cut on a bright rotary microtome (RM2245, Leica) from −0.82 mm bregma to −1.70 mm bregma. A group size of 4–6 animals was evaluated at each time point along with control animals.

Theiler’s Murine Encephalomyelitis Virus Infection and Tissue Processing

For induction of Theiler’s murine encephalomyelitis virus (TMEV) infection, 5-week-old female SJL/JHanHsd mice were intracerebrally infected with 1.63 × 106 PFU/mouse of the BeAn strain of TMEV or mock infected (tissue culture medium) in age-matched controls, as previously described (57, 58). Spinal cords were removed, post-fixed in 10% formalin for 24 hours, decalcified in 25% ethylenediamine tetraacetic acid (EDTA) for 48 hours and embedded in paraffin. For light microscopy, 3-µm transverse sections of the thoracic spinal cord were prepared on a microtome. A group size of 4–6 animals was evaluated at each time point (1 section/animal) (control, 42, 98, 147, 196, and 245 days post-infection [dpi]). Previous studies revealed significant inflammation, as demonstrated by immunohistochemistry targeting CD3 (T-lymphocytes), CD45R (B-lymphocytes) and CD107b (microglia/macrophages), as well as axonal damage in the spinal cord of susceptible SJL-mice during the chronic phase of TME, including the time points 42, 98, 147, 196, and 245 dpi (57, 71).

MS Patient Tissue

Paraffin-embedded postmortem human brain tissue was obtained through a rapid autopsy protocol from 6 patients with different MS progression (ie 3 patients with secondary progressive MS, 2 patients with primary progressive MS, and 1 patient with not-characterized MS) in collaboration with VU-Medical Center, Amsterdam, The Netherlands. MS patients represented both genders (2 female and 4 male MS patients). The ages of the patients ranged from 49 to 80 years. In addition, 3 control brains from patients without evidence of neurological disease or neuropathological alterations were included. This study was carried out accordingly to the applicable national ethical guidelines and legal regulations regarding the use of archival postmortem material.

Immunohistochemistry

For immunohistochemistry, paraffin-embedded sections were dewaxed and heat-unmasked in 10 mM citrate buffer (pH 6.0). The following primary antibodies were used: for myelin, anti-myelin proteolipid protein ([PLP] mouse monoclonal IgG2a, 1:500, Serotec), and -myelin basic protein ([MBP] mouse monoclonal IgG2b, 1:500, Covance); for microglia, anti-ionized calcium-binding adaptor molecule 1 ([Iba-1] rabbit polyclonal IgG, 1:1000, Wako); for neurons, anti-NeuN (mouse monoclonal IgG1, 1:200, Millipore); for identification of oligodendrocytes, anti-myelin-associated neurite outgrowth inhibitor (Nogo-A; rabbit polyclonal IgG, 1:700, Millipore); for astrocytes, anti-glial fibrillary acidic protein ([GFAP] 1:200, rabbit polyclonal IgG, DakoCytomation); for axonal damage, anti-APP (mouse monoclonal IgG1, 1:800, Millipore and rabbit polyclonal IgG, 1:200, Invitrogen); anti-synaptophysin antibodies, (mouse monoclonal IgG1, 1:200, Serotec and 1:200, rabbit monoclonal IgG, YE269, Abcam, Cambridge, UK); anti-neurofilament antibodies, SMI-32, NF-H, non-phosphorylated (mouse monoclonal IgG1, 1:200, Covance), SMI-312, pan NF, phosphorylated (mouse monoclonal IgG1, 1:500, Covance), and NF-M (rabbit polyclonal IgG, 1:300, Abcam). All antibodies were diluted in PBS containing 0.3% Triton-X100. Sections were further incubated with biotinylated secondary antibodies, followed by peroxidase-coupled avidin–biotin complex (ABC Kit, Vector Laboratories, Burlingame, CA). The immunoreaction product was visualized with 3,3′-diaminobenzidine ([DAB] Dako). For immunofluorescent single/double staining, slides were incubated with respective secondary antibodies (Alexa 488 or 555 conjugated goat IgG, 1:500, Invitrogen). Slides were counterstained either with hematoxylin and eosin for DAB or with 4',6-diamidino-2-phenylindole ([DAPI] Invitrogen) for immunofluorescence staining.

Determination of De- and Remyelination

The extent of cuprizone-induced demyelination and initial remyelination was assessed as previously described (56, 59). Sections immunostained for myelin proteins were scored in the midline of the corpus callosum using a light microscope (Olympus BX61) on a scale from 0 (complete demyelination) to 3 (normal myelin); scores 1 and 2 represented the different grades of partial demyelination/remyelination (53, 60). To determinate the extent of demyelination in the thoracic spinal cord of TMEV-infected mice, paraffin sections were stained with PLP for identification of white matter and synaptophysin for mapping grey matter. Demyelinated PLP-negative areas were manually outlined using the Cell’s software® from Olympus BX6. The percentage of demyelination in the thoracic spinal cord segment of TMEV infected mice was determined by calculating the percentage of PLP-negative white matter areas to the total white matter area. The same set of threshold values was used for all animals.

Human material was processed for anti-PLP immunostaining as published previously to stage lesional activity (61). Murine and human control tissue did not display any myelin abnormalities.

Quantification of Synaptophysin- and APP-Positive Spheroids and Glial Reactions

Synaptophysin- and APP-positive spheroids were counted in the central corpus callosum using a magnification of ×400 (Olympus BX61, Cell’s software®) or in the total white matter of thoracic spinal cord sections at a magnification of ×200. The sizes of synaptophysin- and APP-positive spheroids (small [<2 µm], medium [2–7.5 µm], and large [8–15 µm]) were measured using Cell’s software®. The results are presented as a mean ± SEM of spheroid numbers per mm2 (n = 4–6). Quantitation of microglia accumulation was performed for immunoreactivity of Iba-1 staining (62). Data are presented as a number of cells per mm2 (mean ± SEM; n = 4–6). Human material was processed for anti-MHC-II (LN3) staining as published previously to stage lesional activity (61). No evidence of inflammation was detected in age-matched control mice. Sporadic age-related aggregations of activated microglia were observed in single control human samples (data not shown).

Real-Time PCR

Corpus callosum and cerebral cortex were dissected from whole brains under a light microscope. The total RNA was extracted from the tissue using the RNeasy®Mini Kit (Qiagen), as previously described (55, 63). cDNA was synthesized according to the manufacturer’s recommendations using the High Capacity cDNA Reverse Transcription Kit (Life Technologies). Quantitative real-time PCR analysis was performed using the StepOne™ Real-Time PCR System and appropriate TaqMan probes (Life Technologies). The ΔΔCt method was applied to determine differences in the expression of Synaptophysin, IL-1β, TNF-α, Kif1a, Kif1b, Kif1c Kif5a, Kif5b, Kif5c, and dynein genes between cuprizone-treated and age-matched control animals (n = 4–5/group). Changes in mRNA expression levels were calculated after normalization to the arithmetic mean of HPRT-1 and GAPDH. (1) Ct target treated − Ct mean (HPRT and GAPDH) treated = ΔCt treated. (2) Ct target control − Ct mean (HPRT and GAPDH) control = ΔCt control. (3) ΔCt control + (n − 1)ΔCt control + nΔCt control = Σ ΔCt control + (n − 1)ΔCt control + n ΔCt control/n = ΔCt control mean. (4) ΔCt treated − ΔCt control mean = ΔΔCt treated. (5) ΔCt control − ΔCt control mean =ΔΔCt control. (6). Fold change treated = 2−ΔΔCt treated. (7) Fold change control = 2−ΔΔCt control.

Statistical Analysis

Statistical analysis was performed using two-way analysis of variance (ANOVA) or one-way ANOVA followed by the Tukey’s multiple comparison post hoc test. All data are given as arithmetic means ± SE. P values of the ANOVAs are given in the Results section; group comparisons derived from post hoc analysis are provided in the figures. Correlation and linear regression analysis was performed by applying the Pearson correlation test. The normality of all values was evaluated using Kolmogorov–Smirnov test. In all cases, significant effects are indicated by asterisks (compared to the preceding time point) or hash marks (compared to controls) (*#p < 0.05; **##p < 0.01; ***###p < 0.001).

RESULTS

Numbers and Sizes of Synaptophysin-Positive Spheroids Change during Cuprizone-Induced Demyelination

To investigate the distribution of synaptophysin under both physiological and pathological conditions in the CNS, immunohistochemical staining of coronal brain sections from control and cuprizone challenged mice were performed. We used 2 different antibodies for synaptophysin: a mouse monoclonal IgG from Serotec and rabbit monoclonal from Abcam. Both antibodies showed similar staining patterns (

). The grey matter of the cerebrum and cerebellum in controls displayed a widespread fine granular synaptophysin-positive signal (Fig. 1A, B), whereas a few small physiological synaptophysin-positive dots were found in the white matter of the corpus callosum (Fig. 1C).
FIGURE 1

(A–C) Representative sections illustrate immunohistochemical staining for synaptophysin (Syp) in the cerebral cortex and hippocampus (A), cerebellum (B), and corpus callosum (C) of untreated control mice. (D) Accumulation of synaptophysin-positive vesicles = spheroids/ovoids/bulbs in the corpus callosum of a cuprizone-treated mouse. Arrows indicate the different sizes of synaptophysin-positive-spheroids; small [S], <2 µm (as in panel C); middle-sized [M], 2–7.5 µm; large [L], 8–15 µm, pathological size. (E, F) Graphs show numbers of different sized synaptophysin-positive spheroids in the corpus callosum during acute (5 weeks, E) and chronic (12 weeks, F) demyelination followed by remyelination after stopping cuprizone. Significant effects between different time points were calculated separately for each type/size of bulbs (*p <0.05, **p < 0.01, **p < 0.001). Significant effects for different cuprizone time treatments in comparison to the untreated control: #p < 0.05, ##p < 0.01, ###p < 0.001), n = 4–6. (G, H) Representative images show synaptophysin-positive bulbs in the demyelinated hippocampus (G) and internal capsule (H). (I-K) Representative images show that synaptophysin does not colocalize with the oligodendroglial marker Nogo-A (I), the astrocytic marker GFAP (J), or with the microglial marker Iba-1 (K). Scale bar in (K) = 100 µm, applies to (I) and (J).

FIGURE 1

(A–C) Representative sections illustrate immunohistochemical staining for synaptophysin (Syp) in the cerebral cortex and hippocampus (A), cerebellum (B), and corpus callosum (C) of untreated control mice. (D) Accumulation of synaptophysin-positive vesicles = spheroids/ovoids/bulbs in the corpus callosum of a cuprizone-treated mouse. Arrows indicate the different sizes of synaptophysin-positive-spheroids; small [S], <2 µm (as in panel C); middle-sized [M], 2–7.5 µm; large [L], 8–15 µm, pathological size. (E, F) Graphs show numbers of different sized synaptophysin-positive spheroids in the corpus callosum during acute (5 weeks, E) and chronic (12 weeks, F) demyelination followed by remyelination after stopping cuprizone. Significant effects between different time points were calculated separately for each type/size of bulbs (*p <0.05, **p < 0.01, **p < 0.001). Significant effects for different cuprizone time treatments in comparison to the untreated control: #p < 0.05, ##p < 0.01, ###p < 0.001), n = 4–6. (G, H) Representative images show synaptophysin-positive bulbs in the demyelinated hippocampus (G) and internal capsule (H). (I-K) Representative images show that synaptophysin does not colocalize with the oligodendroglial marker Nogo-A (I), the astrocytic marker GFAP (J), or with the microglial marker Iba-1 (K). Scale bar in (K) = 100 µm, applies to (I) and (J).

During cuprizone-induced demyelination, the sizes of synaptophysin-positive dots and their amounts increased (Fig. 1E, F; p < 0.0001 for small and medium-sized spheroids during both an acute and chronic demyelination; p < 0.001 for large spheroids/ovoids in both acute and chronic demyelination). Numbers of small dots decreased significantly during cuprizone-induced demyelination reaching the lowest numbers at the time point week 5 (which corresponds to nearly complete demyelination in the corpus callosum in this model) and remained at a low level during chronic demyelination of 12 weeks. When animals were allowed to remyelinate after 5-week cuprizone treatment, the small synaptophysin spheroids recovered already after 2 weeks of remyelination. In contrast, after chronic demyelination, small physiological synaptophysin-positive spheroids recovered with a delay and a significant increase occurred after 4 weeks of remyelination.

Medium-sized spheroids (2–7.5 µm) were only rarely found in controls. During cuprizone-induced demyelination, the numbers of medium-sized synaptophysin-positive bulbs increased dramatically reaching a peak at the time point week 4, which corresponds to the peak of microglial activation in this model. Thereafter, the numbers of medium-sized spheroids decreased even during chronic demyelination at 12 weeks.

Large synaptophysin-positive spheroids (8–15 µm) were also not found in the corpus callosum in controls. During demyelination, their numbers increased but did not reach significant levels during the short 5-week period of demyelination. During chronic demyelination, significantly, numbers of large spheroids were detected at the time point week 6 and remained increased during the later demyelination phase. During remyelination, large spheroids were still present in low numbers.

Summing up, we hypothesized that small synaptophysin-positive dots represent physiological synaptic vesicles transported through the axonal network, whereas medium-and large-sized synaptophysin-positive bulbs/ovoids/spheroids reflect pathological vesicle accumulations due to axonal transport disturbances. Similar synaptophysin patterns were observed in other prominent white matter tracts such as the internal capsule or hippocampal white matter (Fig. 1G, H). Double staining demonstrated that synaptophysin did not colocalize with the oligodendroglial marker Nogo-A (Fig. 1I), astrocytic marker GFAP (Fig. 1J), or the microglia marker Iba-1 (Fig. 1K).

Synaptophysin as a Marker of Axonal Transport Disturbances in the Cuprizone Model

The next step aimed to verify synaptophysin as a marker to study axonal damage and/or axonal transport disturbances. We performed immunohistochemical double staining for synaptophysin and APP, a widely accepted marker of axonal damage. For quantification, only synaptophysin-positive spheroids >2 µm were considered as pathological vesicles because small synaptophysin-positive dots were considered to be physiological (Figs. 1C,4A, C,5G).

Double staining for APP/synaptophysin revealed that synaptophysin-positive spheroids colocalized with APP during de- and remyelination in the corpus callosum (Fig. 2A–H). However, during the time point of strong microglia activation and demyelination, some APP-positive structures that were not positive for synaptophysin were found. Because it is known that APP may colocalize with reactive astrocytes (64), additional double staining with anti–GFAP was performed. GFAP-positive astrocytes were also found to express APP (Fig. 2I, K, M), whereas there were not GFAP/synaptophysin double-positive cells found (Fig. 2L, N). Thus, for colocalization studies only APP-positive spheroids without nuclear co-staining were counted (Fig. 2A, B, D, G).

FIGURE 2

(A, B) Graphs represent the quantitative analysis of APP, synaptophysin (Syp), and double-positive spheroids in the corpus callosum of cuprizone-treated mice with acute demyelination (A) and chronic demyelination (B). Two-way ANOVA analysis identify that there were no significant differences between numbers of APP- and synaptophysin-positive spheroids during both acute and chronic demyelination or during the remyelination periods. Significant effects between different time points are indicated by asterisks (*p < 0.05, **p < 0.01, **p < 0.001). Significant effects for different cuprizone time treatments in comparison to the untreated control were indicated by hatch marks (#p < 0.05, ##p < 0.01, ###p < 0.001), n = 4–6. (C–H) Representative pictures show colocalization in the corpus callosum of both markers at 4- and 12-week time points. Arrows in (D) and (E) indicate astrocytes expressing APP in the corpus callosum. (I–N) Representative images show that APP (MAB 348) was also expressed in reactive astrocytes in the hippocampus; GFAP and synaptophysin are not colocalized.

FIGURE 2

(A, B) Graphs represent the quantitative analysis of APP, synaptophysin (Syp), and double-positive spheroids in the corpus callosum of cuprizone-treated mice with acute demyelination (A) and chronic demyelination (B). Two-way ANOVA analysis identify that there were no significant differences between numbers of APP- and synaptophysin-positive spheroids during both acute and chronic demyelination or during the remyelination periods. Significant effects between different time points are indicated by asterisks (*p < 0.05, **p < 0.01, **p < 0.001). Significant effects for different cuprizone time treatments in comparison to the untreated control were indicated by hatch marks (#p < 0.05, ##p < 0.01, ###p < 0.001), n = 4–6. (C–H) Representative pictures show colocalization in the corpus callosum of both markers at 4- and 12-week time points. Arrows in (D) and (E) indicate astrocytes expressing APP in the corpus callosum. (I–N) Representative images show that APP (MAB 348) was also expressed in reactive astrocytes in the hippocampus; GFAP and synaptophysin are not colocalized.

Colocalization studies for synaptophysin and SMI-32 (another established marker of axonal damage) were next performed. In untreated control animals neurofilaments were highly phosphorylated, as shown by immunostaining with the marker SMI-312, which stained highly phosphorylated axonal epitopes on neurofilaments M (medium) and H (heavy) (Fig. 3B). Over the course of demyelination, neurofilaments substantially lost their phosphorylation. However, SMI-312-positive swellings/traces could be detected in the affected corpus callosum (Fig. 3D, F, H). SMI-32 visualizes non-phosphorylated neurofilament H. In controls, large axons were not marked with SMI-32 in the corpus callosum (Fig. 3A). Numerous non-phosphorylated SMI-32-positive spheroids were, however, identified at the time point week 4, which corresponded to the peak of microglia activation and strong demyelination (Fig. 3C, E). During early and complete demyelination (weeks 3–5), SMI-32- or SMI-312-positive spheroids only rarely colocalized with synaptophysin (Fig. 3C, 3E, example for colocalization: 3F, F1–F3). In contrast, at later time points beginning at week 6 and further on during chronic demyelination SMI-32 and SMI-312 were predominantly located in large synaptophysin-positive spheroids (Fig. 3G, H); these were also-positive for APP (data not shown). Nevertheless, mainly single synaptophysin-positive spheroids and some single SMI-32 or SMI-312-positive spheroids were detected.

FIGURE 3

Immunohistochemical staining of the corpus callosum evaluating phosphorylation of neurofilaments and their colocalization with synaptophysin (Syp) during cuprizone-induced demyelination. (A–H) SMI-32 detecting non-phosphorylated heavy neurofilaments are shown in panels (A, C, E, G). SMI-312 detecting phosphorylated neurofilaments H and M are illustrated in panels (B, D, F, H). Inserts in (C) and (D) show higher magnification images. Neurofilaments were highly phosphorylated in untreated control animals (B). During the course of demyelination, neurofilaments lost their phosphorylation (D, F, H). Moreover, mostly dephosphorylated neurofilaments (E) but also some phosphorylated neurofilaments (F) cannot be properly transported throughout axons and accumulate in numerous small bulbs. These neurofilament bulbs predominantly did not colocalize with synaptophysin-positive spheroids. However, during the progression of demyelination both markers were found in similar structures, i.e. large ovoids probably representing transected axons (G, H). F1, F2, F3 (from the boxed area in F) also show synaptophysin-positive transected axons.

FIGURE 3

Immunohistochemical staining of the corpus callosum evaluating phosphorylation of neurofilaments and their colocalization with synaptophysin (Syp) during cuprizone-induced demyelination. (A–H) SMI-32 detecting non-phosphorylated heavy neurofilaments are shown in panels (A, C, E, G). SMI-312 detecting phosphorylated neurofilaments H and M are illustrated in panels (B, D, F, H). Inserts in (C) and (D) show higher magnification images. Neurofilaments were highly phosphorylated in untreated control animals (B). During the course of demyelination, neurofilaments lost their phosphorylation (D, F, H). Moreover, mostly dephosphorylated neurofilaments (E) but also some phosphorylated neurofilaments (F) cannot be properly transported throughout axons and accumulate in numerous small bulbs. These neurofilament bulbs predominantly did not colocalize with synaptophysin-positive spheroids. However, during the progression of demyelination both markers were found in similar structures, i.e. large ovoids probably representing transected axons (G, H). F1, F2, F3 (from the boxed area in F) also show synaptophysin-positive transected axons.

The expression and distribution of neurofilament medium chain (neurofilament M) was also analyzed (Fig. 4A–E). In control animals neurofilament M was observed inter alia in cortical neuronal bodies and in callosal axons; physiological synaptophysin-positive vesicles were located inside of neuro-filament-positive axons (Fig. 4A, C). Upon demyelination the intensity of neurofilament M staining decreased and few neurofilament M-positive swellings appeared in the affected corpus callosum (Fig. 4B, D, E). Colocalization studies with synaptophysin (Fig. 4B, D, E), and APP (data not shown) revealed aligned patterns similar to those of SMI-312 and SMI-32.

FIGURE 4

(A-E) Representative images show neurofilament medium chain staining in the corpus callosum of control mice (A, C) and cuprizone-treated mice (B, D, E). Panels (A) and (B) are confocal images.

FIGURE 4

(A-E) Representative images show neurofilament medium chain staining in the corpus callosum of control mice (A, C) and cuprizone-treated mice (B, D, E). Panels (A) and (B) are confocal images.

Axonal Disturbances during Microglia Activation in the Cuprizone Model

To investigate the reasons for the axonal transport disturbances we first investigated the gene expression of different kinesins and dynein, which are important motor proteins responsible for anterograde and retrograde vesicle transport in cortical neurons. Hares et al reported reduced axonal motor protein expression in non-lesional grey matter in MS patients (65). In our study, apart from kinesin 1b, which was upregulated at week 4 of demyelination (

D), we did not identify significant alterations of mRNA expression for either kinesins 1a, 1c, 5a, b, and c nor for dynein in the cortical grey matter at all time points investigated (). Synaptophysin mRNA expression was also not altered in cortical grey matter during the course of acute demyelination ().

We next tested whether myelin loss might be responsible for axonal transport disturbances. Thus, we correlated the callosal expression of myelin proteins MBP and PLP to the appearance of pathological medium-sized and large synap- tophysin-positive axonal swellings in the corpus callosum ((Figs. 5G-L, 6A). There was a positive correlation for MBP during acute demyelination (p = 0.0051; R = 0.8856; r = −0.9410) (Fig. 6A, D), but not for PLP (p = 0.1920; R = 0.3807; r = −0.617) (Fig. 6A, C). For chronic demyelination we did not see any correlation for either MBP (p = 0.0738; R = 0.3867; r = −0.6218) or PLP (p = 0.3164; R = 0.1426; r = −0.3776) (Fig. 6F, G). During severe chronic demyelination the entire amount of pathological synaptophysin-positive spheroids (medium- and large-sized) did not increase (Fig. 6B). Moreover, medium-sized bulbs even decreased in their numbers while the amount of large bulbs rose slightly (Figs. 1F). These large bulbs persisted after 4 weeks of remyelination. During remyelination some synaptophysin-positive bulbs were surrounded by PLP-positive myelin, suggesting that disturbed axons can be remyelinated (Fig. 5O, P). Finally, we investigated the relationship of microglia infiltration/activation and the appearance of medium-and large-sized synaptophysin-positive ovoids. We found a strong correlation between these 2 parameters during both acute and chronic demyelination (acute: p = 0.0188; R = 0.7849; r = 0.8860; chronic: p = 0.0075; R = 0.6635; r = 0.8146) (Figs. 5A–F, M, N, 6A, B, E, H).

(A-P) Representative sections show immunostaining in the corpus callosum for synaptophysin/Iba-1 (A-F) and synaptophysin/PLP (G–L) during acute and chronic cuprizone induced demyelination and subsequent remyelination. Inserts in (A–F) show the morphology/shape of microglia identifying their activation state. Inserts in (G–L) show higher magnification of synaptophysin accumulations. Panels (M) and (N) are confocal images from RCA-1 (microglia activation marker, green) and synaptophysin (red) during inflammation in cuprizone-treated mice. Panels (O) and (P) show remyelinated axons (PLP, green) with internal synaptophysin accumulation (red).

FIGURE 5

(A-P) Representative sections show immunostaining in the corpus callosum for synaptophysin/Iba-1 (A-F) and synaptophysin/PLP (G–L) during acute and chronic cuprizone induced demyelination and subsequent remyelination. Inserts in (A–F) show the morphology/shape of microglia identifying their activation state. Inserts in (G–L) show higher magnification of synaptophysin accumulations. Panels (M) and (N) are confocal images from RCA-1 (microglia activation marker, green) and synaptophysin (red) during inflammation in cuprizone-treated mice. Panels (O) and (P) show remyelinated axons (PLP, green) with internal synaptophysin accumulation (red).

FIGURE 6

The appearance of synaptophysin (Syp)-positive pathological (medium-and large-sized) spheroids was compared and correlated with the extent of demyelination (proteolipid protein: PLP, myelin basic protein: MBP) and microglia infiltration (Iba-1) induced by cuprizone feeding (acute or chronic). (A, B) Graphs show the extent of microglia accumulation, appearance of pathological synaptophysin bulbs and the extent of de- and remyelination as judged by scoring of MBP and PLP immunostaining. Score of 3 represents complete myelination; score of 0 represents complete demyelination. (C–H) Correlation analyses of synaptophysin-positive bulbs with the extent of de- and remyelination and microglia numbers (n = 5–6).

FIGURE 6

The appearance of synaptophysin (Syp)-positive pathological (medium-and large-sized) spheroids was compared and correlated with the extent of demyelination (proteolipid protein: PLP, myelin basic protein: MBP) and microglia infiltration (Iba-1) induced by cuprizone feeding (acute or chronic). (A, B) Graphs show the extent of microglia accumulation, appearance of pathological synaptophysin bulbs and the extent of de- and remyelination as judged by scoring of MBP and PLP immunostaining. Score of 3 represents complete myelination; score of 0 represents complete demyelination. (C–H) Correlation analyses of synaptophysin-positive bulbs with the extent of de- and remyelination and microglia numbers (n = 5–6).

Changes in mRNA expression for the inflammatory cytokines IL-1β and tumor necrosis factor (TNF) were found in the cortex (area of neuronal cell bodies) and the corpus callosum (axonal area). The expression of these cytokines peaked after week 4/5 weeks of cuprizone diet and matched with the maximum of synaptophysin amount (Fig. 7B, C, E, F).

Figure 7

(A, D) Sketches of the mouse brain in coronal section. The marked areas in the cortex and corpus callosum represent investigated regions. (B, C) Graphs indicate mRNA expression of the inflammatory cytokines TNF and IL-1β in the cerebral cortex. (E, F) mRNA expression of TNF and IL-1β in the corpus callosum, Significant effects between different time points are indicated as: *p < 0.05, **p < 0.01, **p < 0.001. Significant effects for different cuprizone time treatments in comparison to the untreated control are indicated as: #p < 0.05, ##p < 0.01, ###p < 0.001), n = 4–5.

Figure 7

(A, D) Sketches of the mouse brain in coronal section. The marked areas in the cortex and corpus callosum represent investigated regions. (B, C) Graphs indicate mRNA expression of the inflammatory cytokines TNF and IL-1β in the cerebral cortex. (E, F) mRNA expression of TNF and IL-1β in the corpus callosum, Significant effects between different time points are indicated as: *p < 0.05, **p < 0.01, **p < 0.001. Significant effects for different cuprizone time treatments in comparison to the untreated control are indicated as: #p < 0.05, ##p < 0.01, ###p < 0.001), n = 4–5.

Thus, because we found a strong correlation between microglia accumulation and synaptophysin-positive spheroid numbers, we conclude that inflammation might be the strongest factor leading to acute axonal transport disturbances. In addition, the amount of synaptophysin aggregations decreased during chronic demyelination, when inflammation abated.

Synaptophysin as a Marker for Axonal Damage in Theiler’s Murine Encephalomyelitis Virus-Induced Demyelination

To confirm synaptophysin as a reliable marker to detect axonal transport damage during demyelinating conditions, a second model of CNS neuroinflammation was studied. Immunohistochemical analyses were performed in thoracic spinal cord sections after TMEV-induced demyelination. Infiltration of microglia/macrophages was visualized using Iba-1 staining. As shown in Figure 8, a scattered accumulation of activated microglia/macrophages could be observed already after 42 dpi. Microglial activation peaked at 98 and 147 dpi with a subsequent decline on days 196 and 245 (p < 0.001) (Fig. 8A). Microglia/macrophage infiltration followed not only a temporal sequence but also a special spatial pattern. First, microglia/macrophages appeared in the ventral part of the spinal cord (42 dpi) and then spread to the lateral parts (98, 147 dpi). At the latest time point analyzed, activated microglia/macrophages were observed predominantly in the dorsal parts between the dorsal horns of the spinal cord (Fig. 8E–H).

FIGURE 8

(A–C) Graphs show microglia/macrophage infiltration (A) and the course of demyelination (B) during Theiler’s virus induced encephalomyelitis in the thoracic segment of murine spinal cord. (C) Panel shows the appearance of pathologic accumulation of synaptophysin (Syp). Significant effects between different time points were indicated as: *p < 0.05, **p < 0.01, **p < 0.001. Significant effects for different time treatments in comparison to the untreated control are indicated as: # p < 0.05, ## p < 0.01, ### p < 0.001), n = 4–6. (D–H) Representative images of thoracic spinal cord segment illustrate infiltration of microglia/macrophage at different days after the injection of Theiler’s murine encephalomyelitis virus. (I–U) Panel (I) shows the appearance of synaptophysin-positive bulbs exclusively in the demyelinated area; (J) and (K) illustrate the appearance of synaptophysin-positive spheroids surrounded by activated microglia/macrophages. (L–N) Colocalization of synaptophysin and APP in the spheroid structures. (O–U) Serial staining of the same spinal cord area during Theiler’s virus induced encephalomyelitis (O, synaptophysin/PLP double staining; P, synaptophysin/Iba-1 double staining; R, synaptophysin/APP double staining; S–U, higher magnification of area shown in R).

FIGURE 8

(A–C) Graphs show microglia/macrophage infiltration (A) and the course of demyelination (B) during Theiler’s virus induced encephalomyelitis in the thoracic segment of murine spinal cord. (C) Panel shows the appearance of pathologic accumulation of synaptophysin (Syp). Significant effects between different time points were indicated as: *p < 0.05, **p < 0.01, **p < 0.001. Significant effects for different time treatments in comparison to the untreated control are indicated as: # p < 0.05, ## p < 0.01, ### p < 0.001), n = 4–6. (D–H) Representative images of thoracic spinal cord segment illustrate infiltration of microglia/macrophage at different days after the injection of Theiler’s murine encephalomyelitis virus. (I–U) Panel (I) shows the appearance of synaptophysin-positive bulbs exclusively in the demyelinated area; (J) and (K) illustrate the appearance of synaptophysin-positive spheroids surrounded by activated microglia/macrophages. (L–N) Colocalization of synaptophysin and APP in the spheroid structures. (O–U) Serial staining of the same spinal cord area during Theiler’s virus induced encephalomyelitis (O, synaptophysin/PLP double staining; P, synaptophysin/Iba-1 double staining; R, synaptophysin/APP double staining; S–U, higher magnification of area shown in R).

The appearance of demyelinating areas showed a similar distribution pattern. Demyelination began at 42 dpi and progressed gradually during the next weeks (p < 0.001). Finally, at 245 dpi, nearly 30% of all spinal cords white matter was demyelinated (Fig. 8B). We detected pathological medium- and large-sized synaptophysin-positive spheroids in the white matter during TME-induced demyelination (p = 0.0022) (Fig. 8C). These synaptophysin-positive formations colocalized with APP-positive spheroids (Fig. 8L–N, S–U), and were predominantly observed in demyelinated (Fig. 8I, O) and microglia-enriched areas (Fig. 8J, K, P). Pathological synaptophysin/APP-positive bulbs were not present in untre- ated animals.

Synaptophysin as a Marker for Axonal Damage in CNS Tissue of MS Patients

Synaptophysin immunostaining revealed a fine granular pattern in synapse rich human grey matter areas (Fig. 9T). Ten chronic active demyelinating lesions from 6 MS patients were analyzed. At the edges of 4 MS lesions we observed numerous synaptophysin-positive spheroids (Fig. 9A–K [lesion 1]; Fig. 9L–O [lesion 2]). These areas were characterized by accumulation of LN3-positive microglia/monocytes and diffuse myelin loss (Fig. 9A, B, I, L–O). Some single synaptophysin-positive spheroids were also found in completely demyelinated areas (data not shown). In both cases, synaptophysin-positive spheroids colocalized with APP-positive bulbs (Fig. 9C-H, J, K, N). Synaptophysin-positive/APP-positive bulbs were not detected in the human control brain tissue.

FIGURE 9

Representative images show the expression of synaptophysin (Syp) in CNS tissue of 2 different patients with MS. (A–H) Different immunohistochemical stainings from a chronic active MS lesion (serial sections). Panels (E) and (F) show the higher magnifications from boxed areas 1 in (C) and (D). Panels (G) and (H) demonstrate higher magnification from boxed areas 2 in (C) and D. (I–K) Representative double immunostaining for synaptophysin and Iba-1 demonstrate microglia accumulation (I) and proving colocalization of APP and synaptophysin (J, K). (L–O) Demyelination/inflammation/axonal damage in another MS lesion. DAB staining in (L) and (M) illustrate the extent of demyelination (PLP) and inflammation (LN3 marks HLA-DR-positive cells: lymphocytes and macrophages) in this MS lesion. Panels N and O show higher magnification of the boxed are from this lesion in (L) and (M) demonstrating double expression of APP and synaptophysin in (N) and double staining of PLP and synaptophysin in (O). (P–U) Corpora amylacea are immunostain-positive for APP (P, S), and NeuN (U), but not synaptophysin (R, T).

FIGURE 9

Representative images show the expression of synaptophysin (Syp) in CNS tissue of 2 different patients with MS. (A–H) Different immunohistochemical stainings from a chronic active MS lesion (serial sections). Panels (E) and (F) show the higher magnifications from boxed areas 1 in (C) and (D). Panels (G) and (H) demonstrate higher magnification from boxed areas 2 in (C) and D. (I–K) Representative double immunostaining for synaptophysin and Iba-1 demonstrate microglia accumulation (I) and proving colocalization of APP and synaptophysin (J, K). (L–O) Demyelination/inflammation/axonal damage in another MS lesion. DAB staining in (L) and (M) illustrate the extent of demyelination (PLP) and inflammation (LN3 marks HLA-DR-positive cells: lymphocytes and macrophages) in this MS lesion. Panels N and O show higher magnification of the boxed are from this lesion in (L) and (M) demonstrating double expression of APP and synaptophysin in (N) and double staining of PLP and synaptophysin in (O). (P–U) Corpora amylacea are immunostain-positive for APP (P, S), and NeuN (U), but not synaptophysin (R, T).

DISCUSSION

Vesicular axonal transport disturbances and axonal damage are hallmarks of numerous neurological disorders, including MS (9, 10). Thus, reliable markers for the detection of axonal transport disturbances are required to study the underlying pathomechanisms. Recently, a calpain-derived alpha-II spectrin N-terminal fragment (SNTF) was identified as a new, highly sensitive marker to study different axonal transport disturbances in traumatic brain injury (66). Synaptophysin presents another possible marker to detect axonal damage under different neuropathological conditions such as MS (67–69). Here, we established synaptophysin, an abundant vesicle membrane protein, as a reliable marker for the detection of axonal damage by performing detailed synaptophysin expression studies in 2 animal models of demyelination and in brain tissue from MS patients. The accumulation of synaptophysin during pathological conditions was compared with the well-established axonal damage markers APP (12, 70, 71) and SMI-32 (21, 72).

APP is synthetized in the neuronal cell soma and undergoes fast axonal transport mediated by conventional kinesin (16, 17, 73). Disruptions of axonal transport or axon dissection lead to accumulation of APP-positive vesicles that can be identified using immunohistochemistry (18, 74). During its transport along the axon, the N-terminus of APP is located inside and the C-terminus outside of the vesicles. Previously, it was reported that antibodies against the C-terminus are even more sensitive to detect the accumulation of APP-positive vesicles at the site of axonal damage (19, 75). Thus, the choice of an appropriate anti-APP antibody seems important because APP becomes post-transcriptionally processed by glycosylation, sulfation, and undergoes a complicated post-translational proteolytic processing (14, 76, 77). Moreover, APP is not exclusively expressed in neurons but may occur in other cell types, including reactive astrocytes (64, 78–80). Different splice variants are known, which are predominantly expressed by neurons (APP-695) or other cell types (APP-751, APP-770, L-APP, and APP-639) (81–84). In our study, we detected APP in some reactive astrocytes during cuprizone-induced demyelination, which is in line with previous publications (64). Unfortunately, in some cases it is difficult to distinguish between an APP-positive damaged axon and APP-positive astrocytic processes without co-staining with an astrocytic marker. Thus, additional markers for axonal damage might be helpful to resolve these difficulties. Like APP, synaptophysin is also moved via the fast axonal transport mostly in an anterograde direction. Here, we found that small synaptophysin-positive dots appear in very high numbers in the normal CNS grey matter; fewer are seen in normal white matter. In the cuprizone-induced demyelination model, the amounts of small physiological synaptophysin dots decreased and larger spheroids, which can be considered as pathological accumulations of synaptic vesicles in damaged axons, were found in the corpus callosum and in other white matter structures. Colocalization studies revealed that these spheroid structures are immunopositive for both synaptophysin and APP. This suggests that the entire fast axonal transport mechanism is impaired during cuprizone-induced demyelination because APP and synaptophysin are known to be moved in different vesicular compartments (50).

Further, we compared the expression pattern of synaptophysin with the marker SMI-32, which detects non-phosphorylated neurofilaments (20–23, 85). In the normal white matter, neurofilaments are mostly phosphorylated whereas in demyelinated axons neurofilaments become dephosphorylated (21, 72, 86). Neurofilaments are moved via slow axonal transport (8, 28, 29). As a consequence of aberrant phosphorylation or transport, neurofilaments accumulate in the affected axons (30, 31, 68, 87). In the present work we detected SMI-32-positive bulbs during the acute microglia activation and demyelination phase in the cuprizone model. At this time, most SMI-32-positive spheroids did not colocalize with synaptophysin or APP. This is probably due to the different transport mechanisms, i.e. slow axonal transport for SMI-32 and fast axonal transport for synaptophysin and APP. A similar result was observed in another study that investigated axonal transport disturbances in immature rat brain injury and detected compacted neurofilament (RM014 immunoreactivity) and APP (31). However, we observed some large double-positive SMI-32/synaptophysin or SMI-32/APP bulbs at later time points of cuprizone-induced demyelination and even during remyelination. We hypothesize that the large SMI-32/APP/synaptophysin-positive bulbs might represent permanent axonal damage or terminally transected axons. In contrast, different medium-sized spheroids, which are either APP/synaptophysin double-positive or SMI-32 single-positive, might detect reversible fast or slow axonal transport impairments, i.e. “vesicle jams”, that can disintegrate when inflammation is resolved. This concept was proposed by Ferguson et al in 1997 (74), and later corroborated by others (88), particularly by the group of Kerschensteiner investigating axonal transport disturbances in experimental autoimmune encephalomyelitis (EAE) using in vivo imaging (89). In our study, the amount of synaptophysin-positive bulbs correlated poorly with the loss of myelin proteins during acute demyelination, as judged by PLP or MBP immunostaining. The mRNA expression of kinesins 1a, 1b, 1c, 5a, 5b, 5c, and dynein, which represent main motor proteins responsible for the transport of synaptic vesicles (90, 91), was also not affected in our study. Thus, we hypothesize that inflammation in particular may be the key factor that impairs axonal transport. This assumption is in line with the correlation between the extent of microglia accumulation and the amount of synaptophysin-positive spheroids in the corpus callosum. Moreover, the appearance of synaptophysin-positive bulbs showed a similar temporal pattern as the mRNA expression of the inflammatory cytokines IL-1β and TNF. Stagi et al also showed that microglial NO significantly inhibited axonal transport of synaptophysin enhanced green fluorescent protein-marked synaptic vesicles in hippocampal neuronal cultures (92).

Kornek et al investigated the distribution of voltage–gated calcium channels in samples from MS patients and animals with EAE. They showed that axonal transport was impaired and the α1b subunit of N-type calcium channels accumulated in dystrophic axons of active demyelinating MS brain lesions and spinal cord lesions in EAE similar to APP (93). Frischer et al performed correlative studies between synaptophysin reactivity and accumulation of reactive T cell and macrophage infiltrates in different MS lesions and provided direct evidence that axonal injury is consistently associated with inflammation (67). Nevertheless, the precise sequence of the degenerative events remains an open and intensively discussed question, i.e. whether axonal damage/axonal transport collapse is caused by inflammation or if it is a consequence of demyelination or both (13, 74, 94–99). In a previous study, we showed less microglial activation after astrocyte depletion using the cuprizone model, which led to delayed myelin debris removal. In those mice, the number of synaptophysin-positive spheroids was not changed, suggesting that microglial accumulation is at least not the sole reason for axonal damage (63).

In the present study, we found some differences between the animal models and MS patient tissue. In contrast to the cuprizone model, only a few large- or medium-sized APP/synaptophysin-positive bulbs were found in TME. The differences in the amount of APP/synaptophysin bulbs are probably due to different pathomechanisms underlying axonal damage in the 2 models. Tsunoda et al postulated that axonal damage can occur in TME even prior to demyelination due to neuronal cell death caused by TMEV followed by Wallerian degeneration (Inside-Out model) (100, 101). Later this hypothesis was refined and proposed as a concept for axonal damage in demyelinating diseases, i.e. a primary event caused by neuronal cell death and Wallerian degeneration evoking an inflammatory cascade and secondary axonal damage or axonopathy (Inside-Out and Outside-In model) (102). Because in our study, we examined particularly the demyelinating phase of TME, we probably detected secondary axonal damage.

Finally, we investigated the expression of synaptophysin and the presence of axonal damage in MS patient CNS tissue. Synaptophysin-positive spheroids were found to be colocalized with APP, supporting the finding that synaptophysin is a marker for axonal damage in the human CNS. However, this is in conflict with the hypothesis of Schirmer et al postulating synaptophysin as a marker for neuronal regeneration after an inflammatory insult (85). However, similar to the work of Frischer et al (67), synaptophysin/APP-positive bulbs were found predominantly at the edges of demyelinating lesions, areas with the highest amount of infiltrating immune cells. This observation supports the hypothesis of an inflammatory origin of axonal damage. However, these synaptophysin/APP-positive spheroids were not always found in MS lesions and their amounts varied in different lesions, suggesting a high complexity or different mechanisms underlying axonal damage. This could also be due to different stages of MS lesion development because it is not possible to obtain the material at defined time points. Furthermore, anti-APP antibodies also detected large spheroid–like structures, which were faintly stained with hematoxylin and were identified as corpora amylacea (Fig. 9P, S). Interestingly, corpora amylacea were also positive for NeuN, suggesting a neuronal origin of these aggregations (Fig. 9U). This observation is in line with some previous studies (103, 104). In contrast to anti-APP, anti-synaptophysin antibodies did not immunostain corpora amylacea (Fig. 9R, T) or reactive astrocytes. However, there was sometimes a diffuse fine granular background staining. Thus, for better interpretation of immunohistochemical staining identifying axonal damage in MS tissue as well as in the spinal cord during TME we suggest applying both anti-APP and -synaptophysin with nuclear co-staining.

In conclusion, we confirm here that synaptophysin is a reliable marker to study axonal damage in the cuprizone model of demyelination (105) since it colocalized with APP and did not cross-react with neither oligodendrocytes, astrocytes, nor microglia. Synaptophysin detected axonal damage in demyelinating human MS lesions and may also serve as a marker for studying axonal damage in the TMEV model.

REFERENCES

1
Hirokawa
N
Nitta
R
Okada
Y.
The mechanisms of kinesin motor motility: lessons from the monomeric motor KIF1A
.
Nat Rev Mol Cell Biol
 
2009
;
10
:
877
84
2
Goldstein
AY
Wang
X
Schwarz
TL.
Axonal transport and the delivery of pre-synaptic components
.
Curr Opin Neurobiol
 
2008
;
18
:
495
503
3
Paschal
BM
Vallee
RB.
Retrograde transport by the microtubule-associated protein MAP 1C
.
Nature
 
1987
;
330
:
181
3
4
Vallee
RB
Williams
JC
Varma
D
, et al.  .
Dynein: An ancient motor protein involved in multiple modes of transport
.
J Neurobiol
 
2004
;
58
:
189
200
5
Roy
S
Zhang
B
Lee
VM
, et al.  .
Axonal transport defects: A common theme in neurodegenerative diseases
.
Acta Neuropathol
 
2005
;
109
:
5
13
6
Roy
S
Winton
MJ
Black
MM
, et al.  .
Rapid and intermittent cotransport of slow component-b proteins
.
J Neurosci
 
2007
;
27
:
3131
8
7
Paus
T
Pesaresi
M
French
L.
White matter as a transport system
.
Neuroscience
 
2014
;
276
:
117
25
8
Brown
A.
Axonal transport of membranous and nonmembranous cargoes: A unified perspective
.
J Cell Biol
 
2003
;
160
:
817
21
9
De Vos
KJ
Grierson
AJ
Ackerley
S
, et al.  .
Role of axonal transport in neurodegenerative diseases
.
Annu Rev Neurosci
 
2008
;
31
:
151
73
10
Millecamps
S
Julien
JP.
Axonal transport deficits and neurodegenerative diseases
.
Nat Rev Neurosci
 
2013
;
14
:
161
76
11
Li
GL
Farooque
M
Holtz
A
, et al.  .
Changes of beta-amyloid precursor protein after compression trauma to the spinal cord: an experimental study in the rat using immunohistochemistry
.
J Neurotrauma
 
1995
;
12
:
269
77
12
Sherriff
FE
Bridges
LR
Sivaloganathan
S.
Early detection of axonal injury after human head trauma using immunocytochemistry for beta-amyloid precursor protein
.
Acta Neuropathol
 
1994
;
87
:
55
62
13
Kuhlmann
T
Lingfeld
G
Bitsch
A
, et al.  .
Acute axonal damage in multiple sclerosis is most extensive in early disease stages and decreases over time
.
Brain
 
2002
;
125
:
2202
12
14
Zheng
H
Koo
EH.
Biology and pathophysiology of the amyloid precursor protein
.
Mol Neurodegener
 
2011
;
6
:
27
15
Dawkins
E
Small
DH.
Insights into the physiological function of the beta-amyloid precursor protein: Beyond Alzheimer's disease
.
J Neurochem
 
2014
;
129
:
756
69
16
Koo
EH
Sisodia
SS
Archer
DR
, et al.  .
Precursor of amyloid protein in Alzheimer disease undergoes fast anterograde axonal transport
.
Proc Natl Acad Sci U S A
 
1990
;
87
:
1561
5
17
Kamal
A
Stokin
GB
Yang
Z
, et al.  .
Axonal transport of amyloid precursor protein is mediated by direct binding to the kinesin light chain subunit of kinesin-I
.
Neuron
 
2000
;
28
:
449
59
18
Sherriff
FE
Bridges
LR
Gentleman
SM
, et al.  .
Markers of axonal injury in post mortem human brain
.
Acta Neuropathol
 
1994
;
88
:
433
9
19
Stone
JR
Singleton
RH
Povlishock
JT.
Antibodies to the C-terminus of the beta-amyloid precursor protein (APP): a site specific marker for the detection of traumatic axonal injury
.
Brain Res
 
2000
;
871
:
288
302
20
Sternberger
LA
Sternberger
NH.
Monoclonal antibodies distinguish phosphorylated and nonphosphorylated forms of neurofilaments in situ
.
Proc Natl Acad Sci U S A
 
1983
;
80
:
6126
30
21
Trapp
BD
Peterson
J
Ransohoff
RM
, et al.  .
Axonal transection in the lesions of multiple sclerosis
.
N Engl J Med
 
1998
;
338
:
278
85
22
Schirmer
L
Antel
JP
Brück
W
, et al.  .
Axonal loss and neurofilament phosphorylation changes accompany lesion development and clinical progression in multiple sclerosis
.
Brain Pathol
 
2011
;
21
:
428
40
23
Dahl
D
Perides
G
Bignami
A.
Axonal regeneration in old multiple sclerosis plaques. Immunohistochemical study with monoclonal antibodies to phosphorylated and non-phosphorylated neurofilament proteins
.
Acta Neuropathol
 
1989
;
79
:
154
9
24
de Waegh
SM
Lee
VM
Brady
ST.
Local modulation of neurofilament phosphorylation, axonal caliber, and slow axonal transport by myelinating Schwann cells
.
Cell
 
1992
;
68
:
451
63
25
Shea
TB
Chan
WK.
Regulation of neurofilament dynamics by phosphorylation
.
Eur J Neurosci
 
2008
;
27
:
1893
901
26
Dale
JM
Garcia
ML.
Neurofilament phosphorylation during development and disease: Which came first, the phosphorylation or the accumulation?
J Amino Acids
 
2012
;
2012
:
382107
27
Dashiell
SM
Tanner
SL
Pant
HC
, et al.  .
Myelin-associated glycoprotein modulates expression and phosphorylation of neuronal cytoskeletal elements and their associated kinases
.
J Neurochem
 
2002
;
81
:
1263
72
28
Wang
L
Ho
CL
Sun
D
, et al.  .
Rapid movement of axonal neurofilaments interrupted by prolonged pauses
.
Nat Cell Biol
 
2000
;
2
:
137
41
29
Roy
S
Coffee
P
Smith
G
, et al.  .
Neurofilaments are transported rapidly but intermittently in axons: implications for slow axonal transport
.
J Neurosci
 
2000
;
20
:
6849
61
30
Siedler
DG
Chuah
MI
Kirkcaldie
MT
, et al.  .
Diffuse axonal injury in brain trauma: Insights from alterations in neurofilaments
.
Front Cell Neurosci
 
2014
;
8
:
429
31
DiLeonardi
AM
Huh
JW
Raghupathi
R.
Impaired axonal transport and neurofilament compaction occur in separate populations of injured axons following diffuse brain injury in the immature rat
.
Brain Res
 
2009
;
1263
:
174
82
32
Petzold
A
Gveric
D
Groves
M
, et al.  .
Phosphorylation and compactness of neurofilaments in multiple sclerosis: Indicators of axonal pathology
.
Exp Neurol
 
2008
;
213
:
326
35
33
Wiedenmann
B
Franke
WW.
Identification and localization of synaptophysin, an integral membrane glycoprotein of Mr 38,000 characteristic of presynaptic vesicles
.
Cell
 
1985
;
41
:
1017
28
34
McMahon
HT
Bolshakov
VY
Janz
R
, et al.  .
Synaptophysin, a major synaptic vesicle protein, is not essential for neurotransmitter release
.
Proc Natl Acad Sci U S A
 
1996
;
93
:
4760
4
35
Sudhof
TC
Lottspeich
F
Greengard
P
, et al.  .
A synaptic vesicle protein with a novel cytoplasmic domain and four transmembrane regions
.
Science
 
1987
;
238
:
1142
4
36
Rehm
H
Wiedenmann
B
Betz
H.
Molecular characterization of synaptophysin, a major calcium-binding protein of the synaptic vesicle membrane
.
EMBO J
 
1986
;
5
:
535
41
37
Thiele
C
Hannah
MJ
Fahrenholz
F.
Cholesterol binds to synaptophysin and is required for biogenesis of synaptic vesicles
.
Nat Cell Biol
 
2000
;
2
:
42
9
38
Valtorta
F
Pennuto
M
Bonanomi
D
, et al.  .
Synaptophysin: leading actor or walk-on role in synaptic vesicle exocytosis?
Bioessays
 
2004
;
26
:
445
53
39
Horikawa
HP
Kneussel
M
El Far
O
, et al.  .
Interaction of synaptophysin with the AP-1 adaptor protein gamma-adaptin
.
Mol Cell Neurosci
 
2002
;
21
:
454
62
40
Calakos
N
Scheller
RH.
Vesicle-associated membrane protein and synaptophysin are associated on the synaptic vesicle
.
J Biol Chem
 
1994
;
269
:
24534
7
41
Galli
T
McPherson
PS
De Camilli
P.
The V0 sector of the V-ATPase, synaptobrevin, and synaptophysin are associated on synaptic vesicles in a Triton X-100-resistant, freeze-thawing sensitive, complex
.
J Biol Chem
 
1996
;
271
:
2193
8
42
Mallozzi
C
D'Amore
C
Camerini
S
, et al.  .
Phosphorylation and nitration of tyrosine residues affect functional properties of Synaptophysin and Dynamin I, two proteins involved in exo-endocytosis of synaptic vesicles
.
Biochim Biophys Acta
 
2013
;
1833
:
110
21
43
Prekeris
R
Terrian
DM.
Brain myosin V is a synaptic vesicle-associated motor protein: Evidence for a Ca2+-dependent interaction with the synaptobrevin-synaptophysin complex
.
J Cell Biol
 
1997
;
137
:
1589
601
44
Ohyama
A
Komiya
Y
Igarashi
M.
Globular tail of myosin-V is bound to vamp/synaptobrevin
.
Biochem Biophys Res Commun
 
2001
;
280
:
988
91
45
Tarsa
L
Goda
Y.
Synaptophysin regulates activity-dependent synapse formation in cultured hippocampal neurons
.
Proc Natl Acad Sci U S A
 
2002
;
99
:
1012
6
46
Gonzalez-Jamett
AM
Baez-Matus
X
Hevia
MA
, et al.  .
The association of dynamin with synaptophysin regulates quantal size and duration of exocytotic events in chromaffin cells
.
J Neurosci
 
2010
;
30
:
10683
91
47
Daly
C
Ziff
EB.
Ca2+-dependent formation of a dynamin-synaptophysin complex: potential role in synaptic vesicle endocytosis
.
J Biol Chem
 
2002
;
277
:
9010
5
48
Daly
C
Sugimori
M
Moreira
JE
, et al.  .
Synaptophysin regulates clathrin-independent endocytosis of synaptic vesicles
.
Proc Natl Acad Sci U S A
 
2000
;
97
:
6120
5
49
Nakata
T
Terada
S
Hirokawa
N.
Visualization of the dynamics of synaptic vesicle and plasma membrane proteins in living axons
.
J Cell Biol
 
1998
;
140
:
659
74
50
Kaether
C
Skehel
P
Dotti
CG.
Axonal membrane proteins are transported in distinct carriers: A two-color video microscopy study in cultured hippocampal neurons
.
Mol Biol Cell
 
2000
;
11
:
1213
24
51
Nicklas
W
Baneux
P
Boot
R
, et al.  .
Recommendations for the health monitoring of rodent and rabbit colonies in breeding and experimental units
.
Lab Anim
 
2002
;
36
:
20
42
52
Skripuletz
T
Bussmann
JH
Gudi
V
, et al.  .
Cerebellar cortical demyelination in the murine cuprizone model
.
Brain Pathol
 
2010
;
20
:
301
12
53
Skripuletz
T
Manzel
A
Gropengiesser
K
, et al.  .
Pivotal role of choline metabolites in remyelination
.
Brain
 
2015
;
138
:
398
413
54
Lindner
M
Fokuhl
J
Linsmeier
F
, et al.  .
Chronic toxic demyelination in the central nervous system leads to axonal damage despite remyelination
.
Neurosci Lett
 
2009
;
453
:
120
5
55
Gudi
V
Skuljec
J
Yildiz
O
, et al.  .
Spatial and temporal profiles of growth factor expression during CNS demyelination reveal the dynamics of repair priming
.
PLoS One
 
2011
;
6
:
e22623
56
Benardais
K
Gudi
V
Gai
L
, et al.  .
Long-term impact of neonatal inflammation on demyelination and remyelination in the central nervous system
.
Glia
 
2014
;
62
:
1659
70
57
Herder
V
Hansmann
F
Stangel
M
, et al.  .
Cuprizone inhibits demyelinating leukomyelitis by reducing immune responses without virus exacerbation in an infectious model of multiple sclerosis
.
J Neuroimmunol
 
2012
;
244
:
84
93
58
Ulrich
R
Baumgartner
W
Gerhauser
I
, et al.  .
MMP-12, MMP-3, and TIMP-1 are markedly upregulated in chronic demyelinating theiler murine encephalomyelitis
.
J Neuropathol Exp Neurol
 
2006
;
65
:
783
93
59
Skripuletz
T
Lindner
M
Kotsiari
A
, et al.  .
Cortical demyelination is prominent in the murine cuprizone model and is strain-dependent
.
Am J Pathol
 
2008
;
172
:
1053
61
60
Gudi
V
Moharregh-Khiabani
D
Skripuletz
T
, et al.  .
Regional differences between grey and white matter in cuprizone induced demyelination
.
Brain Res
 
2009
;
1283
:
127
38
61
Grosse-Veldmann
R
Becker
B
Amor
S
, et al.  .
Lesion expansion in experimental demyelination animal models and multiple sclerosis lesions
.
Molec Neurobiol
 
2016
;
53
:
4905
17
62
Skripuletz
T
Salinas Tejedor
L
, et al.  .
The antiviral drug ganciclovir does not inhibit microglial proliferation and activation
.
Sci Rep
 
2015
;
5
:
14935
63
Skripuletz
T
Hackstette
D
Bauer
K
, et al.  .
Astrocytes regulate myelin clearance through recruitment of microglia during cuprizone-induced demyelination
.
Brain
 
2013
;
136
:
147
67
64
Clarner
T
Buschmann
JP
Beyer
C
, et al.  .
Glial amyloid precursor protein expression is restricted to astrocytes in an experimental toxic model of multiple sclerosis
.
J Mol Neurosci
 
2011
;
43
:
268
74
65
Hares
K
Kemp
K
Rice
C
, et al.  .
Reduced axonal motor protein expression in non-lesional grey matter in multiple sclerosis
.
Mult Scler
 
2014
;
20
:
812
21
66
Johnson
VE
Stewart
W
Weber
MT
, et al.  .
SNTF immunostaining reveals previously undetected axonal pathology in traumatic brain injury
.
Acta Neuropathol
 
2016
;
131
:
115
35
67
Frischer
JM
Bramow
S
Dal-Bianco
A
, et al.  .
The relation between inflammation and neurodegeneration in multiple sclerosis brains
.
Brain
 
2009
;
132
:
1175
89
68
Creed
JA
DiLeonardi
AM
Fox
DP
, et al.  .
Concussive brain trauma in the mouse results in acute cognitive deficits and sustained impairment of axonal function
.
J Neurotrauma
 
2011
;
28
:
547
63
69
Katsuse
O
Iseki
E
Marui
W
, et al.  .
Developmental stages of cortical Lewy bodies and their relation to axonal transport blockage in brains of patients with dementia with Lewy bodies
.
J Neurol Sci
 
2003
;
211
:
29
35
70
Cochran
E
Bacci
B
Chen
Y
, et al.  .
Amyloid precursor protein and ubiquitin immunoreactivity in dystrophic axons is not unique to Alzheimer's disease
.
Am J Pathol
 
1991
;
139
:
485
9
71
Kreutzer
M
Seehusen
F
Kreutzer
R
, et al.  .
Axonopathy is associated with complex axonal transport defects in a model of multiple sclerosis
.
Brain Pathol
 
2012
;
22
:
454
71
72
Herrero-Herranz
E
Pardo
LA
Gold
R
, et al.  .
Pattern of axonal injury in murine myelin oligodendrocyte glycoprotein induced experimental autoimmune encephalomyelitis: Implications for multiple sclerosis
.
Neurobiol Dis
 
2008
;
30
:
162
73
73
Szodorai
A
Kuan
YH
Hunzelmann
S
, et al.  .
APP anterograde transport requires Rab3A GTPase activity for assembly of the transport vesicle
.
J Neurosci
 
2009
;
29
:
14534
44
74
Ferguson
B
Matyszak
MK
Esiri
MM
, et al.  .
Axonal damage in acute multiple sclerosis lesions
.
Brain
 
1997
;
120(Pt 3)
:
393
9
75
Hoshino
S
Kobayashi
S
Furukawa
T
, et al.  .
Multiple immunostaining methods to detect traumatic axonal injury in the rat fluid-percussion brain injury model
.
Neurol Med Chir (Tokyo)
 
2003
;
43
:
165
73
76
Haass
C
Kaether
C
Thinakaran
G
, et al.  .
Trafficking and proteolytic processing of APP
.
Cold Spring Harb Perspect Med
 
2012
;
2
:
a006270
77
Muresan
Z
Muresan
VA.
Phosphorylated, carboxy-terminal fragment of beta-amyloid precursor protein localizes to the splicing factor compartment
.
Hum Mol Genet
 
2004
;
13
:
475
88
78
Gehrmann
J
Banati
RB
Cuzner
ML
, et al.  .
Amyloid precursor protein (APP) expression in multiple sclerosis lesions
.
Glia
 
1995
;
15
:
141
51
79
Banati
RB
Gehrmann
J
Wiessner
C
, et al.  .
Glial expression of the β-amyloid precursor protein (APP) in global ischemia
.
J Cereb Blood Flow Metab
 
1995
;
15
:
647
54
.
80
Zhao
J
O'Connor
T
Vassar
R.
The contribution of activated astrocytes to Abeta production: Implications for Alzheimer's disease pathogenesis
.
J Neuroinflammation
 
2011
;
8
:
150
81
Kang
J
Lemaire
HG
Unterbeck
A
, et al.  .
The precursor of Alzheimer's disease amyloid A4 protein resembles a cell-surface receptor
.
Nature
 
1987
;
325
:
733
6
82
Rohan de Silva
HA
Jen
A
Wickenden
C
, et al.  .
Cell-specific expression of beta-amyloid precursor protein isoform mRNAs and proteins in neurons and astrocytes
.
Brain Res Mol Brain Res
 
1997
;
47
:
147
56
83
Sisodia
SS
Koo
EH
Hoffman
PN
, et al.  .
Identification and transport of full-length amyloid precursor proteins in rat peripheral nervous system
.
J Neurosci
 
1993
;
13
:
3136
42
84
Sola
C
Mengod
G
Probst
A
, et al.  .
Differential regional and cellular distribution of beta-amyloid precursor protein messenger RNAs containing and lacking the Kunitz protease inhibitor domain in the brain of human, rat and mouse
.
Neuroscience
 
1993
;
53
:
267
95
85
Schirmer
L
Merkler
D
Konig
FB
, et al.  .
Neuroaxonal regeneration is more pronounced in early multiple sclerosis than in traumatic brain injury lesions
.
Brain Pathol
 
2013
;
23
:
2
12
86
Brady
ST
Witt
AS
Kirkpatrick
LL
, et al.  .
Formation of compact myelin is required for maturation of the axonal cytoskeleton
.
J Neurosci
 
1999
;
19
:
7278
88
87
Perrot
R
Berges
R
Bocquet
A
, et al.  .
Review of the multiple aspects of neurofilament functions, and their possible contribution to neurodegeneration
.
Mol Neurobiol
 
2008
;
38
:
27
65
88
Mancardi
G
Hart
B
Roccatagliata
L
, et al.  .
Demyelination and axonal damage in a non-human primate model of multiple sclerosis
.
J. Neurol. Sci
 
2001
;
184
:
41
9
89
Nikic
I
Merkler
D
Sorbara
C
, et al.  .
A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis
.
Nat Med
 
2011
;
17
:
495
9
90
Hirokawa
N
Noda
Y
Tanaka
Y
, et al.  .
Kinesin superfamily motor proteins and intracellular transport
.
Nat Rev Mol Cell Biol
 
2009
;
10
:
682
96
91
Hirokawa
N
Noda
Y.
Intracellular transport and kinesin superfamily proteins, KIFs: Structure, function, and dynamics
.
Physiol Rev
 
2008
;
88
:
1089
118
92
Stagi
M
Dittrich
PS
Frank
N
, et al.  .
Breakdown of axonal synaptic vesicle precursor transport by microglial nitric oxide
.
J Neurosci
 
2005
;
25
:
352
62
93
Kornek
B
Storch
MK
Bauer
J
, et al.  .
Distribution of a calcium channel subunit in dystrophic axons in multiple sclerosis and experimental autoimmune encephalomyelitis
.
Brain
 
2001
;
124
:
1114
24
94
Bitsch
A
Schuchardt
J
Bunkowski
S
, et al.  .
Acute axonal injury in multiple sclerosis. Correlation with demyelination and inflammation
.
Brain
 
2000
;
123(Pt 6)
:
1174
83
95
Centonze
D
Muzio
L
Rossi
S
, et al.  .
The link between inflammation, synaptic transmission and neurodegeneration in multiple sclerosis
.
Cell Death Differ
 
2010
;
17
:
1083
91
96
Siffrin
V
Vogt
J
Radbruch
H
, et al.  .
Multiple sclerosis—Candidate mechanisms underlying CNS atrophy
.
Trends Neurosci
 
2010
;
33
:
202
10
97
Zindler
E
Zipp
F.
Neuronal injury in chronic CNS inflammation
.
Best Pract Res Clin Anaesthesiol
 
2010
;
24
:
551
62
98
Kornek
B
Storch
MK
Weissert
R
, et al.  .
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
76
99
Neumann
H.
Molecular mechanisms of axonal damage in inflammatory central nervous system diseases
.
Curr Opin Neurol
 
2003
;
16
:
267
73
100
Tsunoda
I
Kuang
LQ
Libbey
JE
, et al.  .
Axonal injury heralds virus-induced demyelination
.
Am J Pathol
 
2003
;
162
:
1259
69
101
Tsunoda
I
Fujinami
RS.
Inside-out versus outside-in models for virus induced demyelination: Axonal damage triggering demyelination
.
Springer Semin Immunopathol
 
2002
;
24
:
105
25
102
Sato
F
Tanaka
H
Hasanovic
F
, et al.  .
Theiler's virus infection: Pathophysiology of demyelination and neurodegeneration
.
Pathophysiology
 
2011
;
18
:
31
41
103
Tate-Ostroff
B
Majocha
RE
Marotta
CA.
Identification of cellular and extracellular sites of amyloid precursor protein extracytoplasmic domain in normal and Alzheimer disease brains
.
Proc Natl Acad Sci U S A
 
1989
;
86
:
745
9
104
Korzhevskii
DE
Giliarov
AV.
Demonstration of nuclear protein neun in the human brain corpora amylacea
.
Morfologiia
 
2007
;
131
:
75
6
105
Gudi
V
Gingele
S
Skripuletz
T
, et al.  .
Glial response during cuprizone-induced de- and remyelination in the CNS: Lessons learned. (Review)
.
Front Cell Neurosci
 
2014
;
8
:
73

Author notes

Viktoria Gudi and Lijie Gai equally contributed to the work as first authors; Martin Stangel and Thomas Skripuletz contributed equally as senior authors. This work is part of Lijie Gai's MD thesis.
The authors declare that they have no competing interests.

Supplementary data