J Neural Transm (1996) 103:1317-1329 _Journal of_ Neural Transmission Springer-Verlag 1996 Printed in Austria Synaptophysin in spinal anterior horn in aging and ALS: an immunohistological study F. F. Cruz-Sfinchez 1, A. MoraU, M. L. Rossi 2, L. Quint@, C. Castej6n ~, E. Tolosa 1, and J. de Belleroche 4 Neurological Tissue Bank, Hospital Clinic-University of Barcelona, Spain 2 Walton Centre for Neurology and Neurosurgery, Liverpool, United Kingdom 3Epidemiology and Biostatistics Unit, Hospital Clinic-University of Barcelona, Spain 4Department of Biochemistry, Chafing Cross Hospital, London, United Kingdom Accepted July 17, 1996 Summary. Aged-related spinal cord changes such as neuronal loss have been related to the degree of clinical severity of amyotrophic lateral sclerosis (ALS); morphological data on synapses are, however, wanting. Variations in synaptophysin (Sph) expression in aging and ALS were thus studied at the level of lower motor neurons in 40 controls with non-neurological diseases and 11 cases of ALS. Control sections of formalin fixed paraffin embedded cervical (C7/8), thoracic (T10) and lumbar spinal cord (L5) and C6, C7, C8 and L5 of ALS cases were stained with haematoxylin and eosin, luxol fast blue (LFB), and immunostained with a mouse monoclonal antibody against Sph. The neuropil of the anterior horn (AH) in all control cases demonstrated Sph positivity. A dot-like pattern of positivity of presynaptic terminals on soma of motor neurons and fine immunoreactivity along neuronal processes were observed. A significant reduction of Sph immunostaining was observed in the neuropil with increasing age and 3 different somatic patterns were seen: a- well preserved Sph reactivity around the soma and the proximal dendrites of histologically normal neurons; b- few chromatolytic neurons showing large numbers of dot-like presynaptic terminals around the cell body and in a "fused" pattern; c- intense, diffuse, and homogeneous reactivity of some neurons. Attenuation of Sph reactivity in the AH neuropil, to its complete loss, was observed in all ALS cases. In addition to patterns a-c, two additional microscopic findings were noted in ALS: d- chromatolytic neurons showing complete absence of Sph reactivity; e- absence of Sph reactivity around the soma and the proximal dendrites of histologically normal surviving neurons. Our findings demonstrate that there is a decrease in Sph immunostaining with aging, thus suggesting an alteration in dendritic networks of the AH with aging. Changes in the pattern of Sph immunoreactivity in cell bodies may
Transcript
J Neural Transm (1996) 103:1317-1329 _Journal o f _
Neural Transmission
�9 Springer-Verlag 1996 Printed in Austria
Synaptophysin in spinal anterior horn in aging and ALS:
an immunohistological study
F. F. Cruz-Sfinchez 1, A. MoraU, M. L. Rossi 2, L. Quint@, C. Castej6n ~, E. Tolosa 1, and J. de Belleroche 4
Neurological Tissue Bank, Hospital Clinic-University of Barcelona, Spain 2 Walton Centre for Neurology and Neurosurgery, Liverpool, United Kingdom
3Epidemiology and Biostatistics Unit, Hospital Clinic-University of Barcelona, Spain 4Department of Biochemistry, Chafing Cross Hospital, London, United Kingdom
Accepted July 17, 1996
Summary. Aged-related spinal cord changes such as neuronal loss have been related to the degree of clinical severity of amyotrophic lateral sclerosis (ALS); morphological data on synapses are, however, wanting. Variations in synaptophysin (Sph) expression in aging and ALS were thus studied at the level of lower motor neurons in 40 controls with non-neurological diseases and 11 cases of ALS. Control sections of formalin fixed paraffin embedded cervical (C7/8), thoracic (T10) and lumbar spinal cord (L5) and C6, C7, C8 and L5 of ALS cases were stained with haematoxylin and eosin, luxol fast blue (LFB), and immunostained with a mouse monoclonal antibody against Sph. The neuropil of the anterior horn (AH) in all control cases demonstrated Sph positivity. A dot-like pattern of positivity of presynaptic terminals on soma of motor neurons and fine immunoreactivity along neuronal processes were observed. A significant reduction of Sph immunostaining was observed in the neuropil with increasing age and 3 different somatic patterns were seen: a- well preserved Sph reactivity around the soma and the proximal dendrites of histologically normal neurons; b- few chromatolytic neurons showing large numbers of dot-like presynaptic terminals around the cell body and in a "fused" pattern; c- intense, diffuse, and homogeneous reactivity of some neurons. Attenuation of Sph reactivity in the AH neuropil, to its complete loss, was observed in all ALS cases. In addition to patterns a-c, two additional microscopic findings were noted in ALS: d- chromatolytic neurons showing complete absence of Sph reactivity; e- absence of Sph reactivity around the soma and the proximal dendrites of histologically normal surviving neurons.
Our findings demonstrate that there is a decrease in Sph immunostaining with aging, thus suggesting an alteration in dendritic networks of the AH with aging. Changes in the pattern of Sph immunoreactivity in cell bodies may
1318 F.F. Cruz-Sfinchez et al.
represent synaptic plasticity and/or degeneration. Reinnervation may also be a possible mechanism as a response to neuronal loss in oldest control cases. Sph reactivity results may thus lend support to the presence of superimposed aging components in ALS cases which may give an insight into explaining the increasing severity of the disease which is encountered with advancing age.
Neuronal loss at the level of the spinal cord is a feature of aging, this finding being supported by electrophysiological (Barnes, 1994) and histological stud- ies (Moral et al., 1994). Motor neuron disease (MND) includes a variety of conditions such as ALS (Rossi, 1994). Some authors have reported on the severe involvement of the dendritic network and dendritic spines in motor neurons of the spinal cord (Kato et al., 1987) and of the neocortex of ALS cases (Ferrer et al., 1991; Rossi et al., 1992).
The neuropil can be seen as the site where dendrites of anterior horn neurons and interneurons synapse with presynaptic terminals of afferent fibres (Ikemoto et al., 1994). In the neuropil of the anterior horn the axo- dendritic synapse is more common than other types of synapse (Peters et al., 1991). The loss of interneurons in ALS (Tsukagoshi et al., 1979; Kato et al., 1987a,b) may also contribute to the alteration of presynaptic terminals. A similar phenomenon could also occur in aging.
In humans, corticospinal axons terminate on the anterior horn motor neurons either on the soma or on dendritic processes (Schoen, 1964; Iwatsubo et al., 1990). ALS cases also show marked corticospinal tracts degeneration (Rossi, 1994) which may be the cause of the severe dendritic network damage.
Synaptophysin (Sph) is a protein obtained from synaptic vesicles (Wiedenmann and Franke, 1985). Antibodies to Sph have proven valuable in the recognition of interneuronal connections. Recently, some authors re- ported findings on Sph reactivity in upper and lower motor neuron diseases (Kawanami et al., 1993; Sasaki et al., 1994; Ikemoto et al., 1994), similar studies in aging are however wanting. Aging mechanisms have also been implicated in determining the severity of ALS (Eisen et al., 1993). For some authors ALS may be caused by "environmental" damage to motor neurons which remains subclinical for several decades but for the concomitant age- related neuronal attrition (Caine et al., 1986). Motor neuron loss has been reported with aging (Tomlinson and Irving, 1977; Moral et al., 1994), however, other changes such as those appertaining to synaptic activity have not previ- ously been reported in aging.
We present a large series of ALS and control cases in an attempt to substantiate whether there are abnormalities in the expression of Sph at the level of lower motor neurons in different age groups, and vs. ALS cases within the same age brackets and to recognize possible aging effects in relation to ALS.
Synaptophysin immunoreactivity in spinal motor neurons 1319
Patients, material and methods
Forty non-neurological controls (Table 1) and 11 cases of ALS were studied (Table 2). Controls included 21 males and 19 females (mean age 60 years, with a range of 20-101 years) which were separated according to age at 10 yearly intervals. ALS cases (Table 2) included 8 males and 3 females (mean age 61.1 years with a range of 43-70). The mean duration of evolution was 38.6 months (range 20-72). Although, terminally, all cases had generalized involvement of the spinal cord, we decided to study C6, C7, C8 and L5 levels in ALS cases.
Table 1. Epidemiological data of controls
Age group No cases Mean age Male/fern Cause of death years years
20-29 5 25,8 2/3 CH Sepsis Traumatism(3)
30-39 5 35,4 3/2 CH CF GITH(2) PTE
40-49 5 45,2 4/1 Sepsis(2) Pneumonia(2) GITH
50-59 4 55,5 2/2 MI(2) Sepsis APO
60-69 6 66,8 3/3 MI(4) CF CH
70-79 6 72,5 3/3 Pneumonia(2) NHL Metastasic Carcinoma CF Sepsis
80-89 5 84,8 1/4 PTE CF Pneumonia CF GITH
90-101 4 95 3/1 GITH MI PTE RF
Total 40 60 21/19
A P O Acute pulmonary oedema; CF Cardiac Failure; CH Cerebral haemorrhage; MI Myocardical infarction; G I T H Gastrointestinal haemorrage; N H L Non-Hodgking lymphoma; P T E Pulmonary tromboembolism; R F Renal Failure. In brackets: number of patients
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Table 2. Clinical data of ALS patients
Age s Sex Cause of death Duration Initial of ALS: symptoms
66 M Pneumonia 38 PPA 70 M Pneumonia 41 DPA 66 M Sepsis 40 DPL 68 M Pneumonia 20 DPA 65 M MI 27 PPL 70 F Pneumonia 24 DPA 47 M Peritonitis 47 DPL 61 F PTE 51 DPL 51 M APO 25 DPL 43 M Pneumonia 40 PPA 66 F Sepsis 72 DPA 61,1A 11M/9F 38,6 B
1Age at death (years). 2Evolution of ALS (months). aMean age of the population (years). BMean evolution of the disease (months). A P O Acute pulmonary oedema; M I Myocardical infarction; D P A Distal paresis of arm; D P L Distal paresis of leg; P P A Proximal paresis of arm; P P L Proximal paresis of leg; P T E Pulmonary tromboembolism; M male; F female
The mean post mortem delay for both groups of cases (controls and ALS) was 9 hours with a range of 2-18 hours. Brain and spinal cord tissue were processed according to a brain banking protocol previously described (Cruz-Sfinchez et al., 1993). Tissue used for the study was fixed in a 10% formalin solution for 2 weeks. Alternate paraffin sections of spinal cord controls at cervical (C7-8), thoracic (T10) and lumbar level (L5) were stained with haematoxylin and eosin, luxol fast blue (LFB), Nissl, and immunostained with a mouse monoclonal antibody against Sph (Dako 1:200) using the avidin-biotin immunoperoxydase complex method (Cruz-Sfinchez et al., 1991). The Marchi impregna- tion technique was used to demonstrate "recent" tract degeneration (Smith, 1960). The same techniques were used on C6, C7, C8 and L5 levels in each ALS patient.
In the attempt to estimate the immunoreactivity for Sph we semiquantitated the intensity of immunostaining (at • magnification): 0 was no staining, 1 scanty (very scarce), 2 mild, 3 moderate and 4 was intense immunoreactivity to Sph. Results were statistically analyzed with the SSPS program. In order to facilitate statistical analysis of data and to valorate variations in immunostaining according to age, we regrouped patients in two age groups: A) 20 to 69 years, B) 69 to 101 years. We then calculated the mean value of intensity and the standard deviation from the mean of each group and level studied. We then applied the U-Mann-Whitney test to compare age groups and the Friedman test for spinal levels.
The same approach to semiquantitation was followed for ALS cases but the intensity of immunostaining was so slight that it precluded any measurement.
Fig. 1. Sph immunoreactivity of a large motor neuron in the spinal cord from a 32 years old man. Few large immunoreactive dots (arrows) on the surface of cell bodies and proximal portion of dendrites and numerous fine dots on the surface of distal portions of
dendrites • Fig. 3. A large motor neuron in the spinal cord from a 80 years old man showing
numerous fine dots on the surface of the cell body. •
Synaptophysin immunoreactivity in spinal motor neurons 1321
1322 F.F. Cruz-Sfinchez et al.: Synaptophysin immunoreactivity
Results
Controls
The grey matter of the anterior horn of control cases showed strong Sph positivity in the neuropil. At high magnification, a dot-like pattern of positiv- ity of presynaptic terminals on soma of anterior horn neurons (Fig. 1) and fine immunoreactivity along neuronal processes were observed. A reduction of neuropil immunostaining (Fig. 2) and an increase in the number of positive synaptic boutons (Fig. 3) on the somatic membrane of some motor neurons were observed in cases aged over 75 years. In these cases, dendritic processes were few and some of them did not show synaptic boutons especially in secondary branches. Primary branches of some neurons also showed a large number of synaptic boutons. There were no statistically significant differences in Sph immunoreactivity between the three levels in the same age group (Table 3), but the differences became highly significative when we compared group A vs B (Table 4). Results support a uniform decrease of Sph immunore- activity in the elderly spinal cord.
Three different patterns of immunostaining were found in the neurons and their processes in cases aged over 40 years (Table 5):
a. well preserved Sph reactivity around the soma and the proximal den- drites of histologically normal neurons in all cases
b. few chromatolytic neurons showing large numbers of dot-like presyn- aptic terminals around the cell body and in a "fused" pattern in 9/30 patients and in none in patients under 40
c. intense, diffuse and homogeneous reactivity of some neurons in 10 of 30 cases, and again in none of patients under 40.
No corticospinal degeneration was observed on LFB stain and with the Marchi impregnation.
Amyotrophic lateral sclerosis
Attenuation of Sph reactivity in the anterior horn neuropil, to its complete loss, was observed in all ALS cases. There were 5 different patterns of neu- ronal Sph immunoreactivity (Table 5):
a. well preserved Sph reactivity around the soma and the proximal den- drites of histologically normal neurons in 10/11 patients
b. chromatolytic neurons showing a large number of dot-like presynaptic terminals around the cell body and in a "fused" pattern (Fig. 4A) in 6/11 patients
Fig. 2. Sph immunoreactivity in the gray matter of hemisections of the lumbar segments of the spinal cord from 4 control cases showing a different degree of the immunostaining. In a 43 years old man (A) Sph immunoreactivity is distributed throughout the entire gray matter. The immunostaining appears to be slightly (in a 62 years old woman) (B), moderately (in a 80 years old man) (C) and strikingly (in a 93 years old man)
(D) decreased according to increasing age
1324 F. F. Cruz-S/mchez et al.
Table 3. Quantitation of Sph immunoreactivity in the three spinal levels studied in the same age group (Friedman test)
Table 4. Quantitation of Sph immunoreactivity in the three spinal levels studied and comparison of results according to the age grouping (U Mann-Whitney test)
Sph synaptophysin. * Number of cases and percentage of neurons that present each pattern in each group. A well preserved Sph reactivity around the soma and the proximal dendrites of histologically normal neurons. B Chromatolytic neurons showing a large number of dot-like presynaptic terminals around the cell body and in a "fused" pattern, C Intense, diffuse and homogeneous reactivity of some neurons. D Chromatolytic neurons showing complete absence of Sph reactivity. E Absence of Sph reactivity around the soma and the proximal dendrites of histologically normal surviving neurons
Synaptophysin immunoreactivity in spinal motor neurons 1325
c. intense, diffuse and homogeneous reactivity of surviving neurons (Fig. 4B) in 5/11 patients
d. chromatolytic neurons showing complete absence of Sph reactivity (Fig. 4C,D) in 8/11 patients and in none of disease free controls
e. absence of Sph reactivity around the soma and the proximal dendrites of histologically normal surviving neurons (Fig. 4E) in 9/11 patients and in none of disease free controls. Very few chromatolytic neurons showing few synaptic boutons (Fig. 4F) were also observed in all cases.
ALS cases showed degeneration of the corticospinal tracts on conventional stains but in 2 cases the Marchi technique was the only one which demon- strated such degeneration.
Discussion
Our study was undertaken to ascertain whether there was a difference in the pattern of Sph immunoreactivity in spinal motor neurons in normal aging and in ALS. In relation to ALS, our results are similar to those previously reported. However, the detailed study of different staining patterns may throw a new light on the degenerating process of motor neurons in aging and ALS.
Several immunohistological studies have found that technical characteris- tics as well as patient-related factors introduce a huge variation and systematic errors in the estimation of synaptophysin immunoreactivity (Masliah et al., 1991; Ince et al., 1995). Therefore, collecting post-mortem human brain samples for research should include accurate matching for ante- and post mortem factors (Cruz-Sfinchez et al., 1993; Ravid et al., 1995). Due to these variabilities, material used in the present study has been collected according to a standardized protocol for sampling, dissection, tissue preparation and accurate matching based on previously published results (Ravid and Winblad, 1993; Ince et al., 1995; Ravid et al., 1995) and our own experience (Cruz- Sfinchez et al., 1993; Palacin et al., 1993).
Results support a uniform decrease in Sph neuropil immunoreactivity as age advances. The decrease in Sph also begins in the spinal cord in the eighth decade of life when CNS plasticity lessens. Kato and co-workers (Kato et al., 1987) demonstrated thinning of dendritic trunks and shrinkage of dendritic trees in ALS. Several authors have also demonstrated morphological changes of dendrites and loss of dendritic spines with aging (Scheibel et al., 1975, 1976, 1977; Coleman and Flood, 1987), therefore, changes affecting dendritic shape or numbers are factors which could contribute to the reduction of Sph immunoreactivity in the neuropil. Damage to presynaptic connections with anterior horn interneurons may be the cause of such abnormalities (Ikemoto et al., 1994).
The presence of specific patterns of immunostaining supports that the alteration of the dendritic network (also reflected by the decrease of Sph immunostaining), is important in the aging process besides neuronal loss. However, the differential patterns of immunoreactivity we found may be related to diverse physiopathological mechanisms.
1326 F.F. Cruz-Sfinchez et al.
Corticospinal tract degeneration was recognized in our ALS cases, which could explain a decrease in afferent fibers and a decrease in presynaptic terminals on dendritic processes which constitute the anterior horn neuropil. However, pyramidal tract degeneration was not observed in controls, whilst a decrease of Sph immunoreactivity was still found in the neuropil thus suggest- ing that the severity of the degenerative changes of spinal motor neurons and in the neuropil is crucial for the reduction of Sph immunoreactivity.
The pattern of immunostaining varied in an age-related and disease- related fashion. In agreement with other authors (Kawanami et al., 1993), surviving motor neurons in ALS showed dense accumulation of immunoreac- tive boutons on the surface of the cell body which may be due to plasticity mechanisms adapting to loss of presynaptic terminals (Sasaki and Maruyama, 1994; Ince et al., 1995). On the contrary, other histologically normal neurons in ALS cases did not show any immunoreaction for Sph. Degeneration of pyramidal tracts may be responsible for the reduction or complete absence of Sph immunoreactivity in most spinal motor neurons in ALS. However, ac- cording to other authors (Ikemoto et al., 1994) elderly controls display an abundance of dots on the remainder of the neuronal surface and the loss of presynaptic terminals appears to be independent of the integrity of the corti- cal tract (Ince et al., 1995). The increased number of dots may be a presynaptic compensatory mechanism for a reduction in synaptic space which, in turn, may be caused by changes in dendrites or neuronal loss. These findings have been elicited in other cerebral areas in aging (Adams, 1987) and in experimen- tal animal models (Hillman and Chen, 1985).
In oldest controls and ALS patients some neurons with chromatolytic appearance and/or atrophy due to loss of proximal processes (Hirano, 1982) showed an increase in Sph immunostaining which may represent another compensatory mechanism for the decrease of synaptic surface. According to Ikemoto and co-workers (Ikemoto et al., 1994) these features may represent synaptic plasticity or a synaptic rearrangement associated with the enlarge- ment of presynaptic terminals and dendritic degeneration. The absence of corticospinal tract degeneration in oldest controls may be explained in terms of a mechanism of reinnervation of some histologically normal remaining neurons as a response to neuronal loss which has been demonstrated by other authors in aging (Tomlinson and Irving, 1977; Sasaki and Maruyama, 1994; Ince et al., 1995).
Several studies demonstrated the relationship of presynaptic abnormali- ties and plasticity mechanisms with abnormal distribution of Ca ++ channel
Fig. 4. Large motor neurons in the spinal cord from ALS patients showing different somatic patterns of immunostaining. A Chromatolytic neurons showing a "fused" pattern of fine dots on the cell surface. B Intense, diffuse and homogeneous reactivity of a surviving neuron. C and D Chromatolytic neurons showing complete absence of imunoreactivity. E Absence of immunoreactivity around the soma and the proximal dendritic portion of a normal-looking surviving neurons. F Chromatolytic neuron show- ing few fine dots on the cell surface (Sph A • B • C • D • E •
F x600)
Synaptophysin immunoreactivity in spinal motor neurons 1327
1328 F.F. Cruz-S~inchez et al.
proteins such as calbindin (Ince et al., 1995) or of g lu tamate receptors (Shaw et al., 1991; Shaw, 1994). The methodologica l approach adop ted in the present s tudy does not allow for correlat ion with these results. However , fur ther studies along this line may contr ibute to elucidate mechanisms related to synaptic abnormali t ies in aging and ALS.
Acknowledgements
Study supported by the BIOMED-1 program (PL931359) from the Commission of the European Communities (EU) and by grants 94/0996 and 96/1320 from the "Fondo de Investigaci6n Sanitaria", Spanish Health Ministry. We are very grateful to Dr. A. Hirano for his helpful advice in the preparation of the manuscript.
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Authors' address: F. F. Cruz-Sfinchez, MD, Banco de Tejidos Neuroldgicos, Servicio de Neurologia, Hospital Clfnico, c/Villarroel 170, E-08036 Barcelona, Spain
