Acta Neuropathol (2004) 107 : 359–364 DOI 10.1007/s00401-004-0821-7
R E G U L A R PA P E R
Toshiya Nakano · Kazuhiro Nakaso · Kenji Nakashima · Eisaku Ohama
Expression of ubiquitin-binding protein p62 in ubiquitin-immunoreactive intraneuronal inclusions in amyotrophic lateral sclerosis with dementia: analysis of five autopsy cases with broad clinicopathological spectrum Received: 10 October 2003 / Revised: 21 December 2003 / Accepted: 30 December 2003 / Published online: 5 February 2004 © Springer-Verlag 2004
Abstract Amyotrophic lateral sclerosis with dementia (ALSD), corresponding to the motor neuron disease type of frontotemporal dementia, is neuropathologically characterized by depletion of the motor neurons, degeneration of the extra-motor cerebral cortices and formation of ubiquitin-immunoreactive (not argyrophilic, tau-negative, α-synuclein-negative) intraneuronal inclusions. Recently, immunoreactivity for ubiquitin-binding protein p62 has been reported in several ubiquitin-containing intraneuronal or intraglial inclusions (e.g. neurofibrillary tangles, Pick bodies, Lewy bodies, glial cytoplasmic inclusions) in various neurodegenerative diseases. We examined p62 immunoreactivity in ubiquitin-immunoreactive intraneuronal inclusions in five ALSD cases with a broad clinicopathological spectrum. p62 immunoreactivity in ubiquitin-immunoreactive intraneuronal inclusions was seen in all cases. The mean proportion of p62-immunoreactive inclusions to the total number of ubiquitin-immunoreactive inclusions (p62/Ub ratio) in the dentate gyrus was 27.5±16.6% (range 6.3–47.3%). There was no correlation between p62/Ub ratio and the severity of dementia, duration of illness or neuropathological severity. Although the main constituent of these inclusions is unknown, our study suggests that p62 contributes to the formation of the inclusions via the same mechanism as in other previously reported neurodegenerative diseases. Since p62 is believed to have a neuroprotective role, the formation of these inclusions may represent a non-harmful, rather protective effect against the neuronal degeneration in ALSD.
T. Nakano (✉) · K. Nakaso · K. Nakashima Department of Neurology, Institute of Neurological Sciences, Faculty of Medicine, Tottori University, 36-1 Nishi-cho, 683-8504 Yonago, Japan Tel.: +81-859-348032, Fax: +81-859-348083, e-mail:
[email protected] E. Ohama Department of Neuropathology, Institute of Neurological Sciences, Faculty of Medicine, Tottori University, 36-1 Nishi-cho, 683-8504 Yonago, Japan
Keywords p62 · Ubiquitin · Ubiquitin-binding protein · Ubiquitin-immunoreactive intraneuronal inclusion · Amyotrophic lateral sclerosis with dementia
Introduction Amyotrophic lateral sclerosis with dementia (ALSD, YuasaMitsuyama disease), corresponding to a motor neuron disease type of frontotemporal dementia, is neuropathologically characterized by depletion of the motor neurons, occurrence of Bunina bodies in the remaining motor neurons, degeneration of the extra-motor cerebral cortices and formation of ubiquitin-immunoreactive (not argyrophilic, tau-negative, α-synuclein-negative) intraneuronal inclusions [2, 5, 6, 10, 11, 13, 14, 15, 16, 22, 25, 26]. However, the main constituent of these inclusions is still unknown. Therefore, the mechanism of inclusion formation and the significance of inclusions in the disease process are unclear. p62 is a cytosolic 62-kDa protein, initially identified as a binding protein to the Src homology 2 (SH2) domain of p56lck in a phosphotyrosine-independent manner [3, 17]. Recently, it has been reported that p62 also binds noncovalently to ubiquitin, suggesting an important role in the ubiquitin pathway [20, 23]. Furthermore, p62 immunoreactivity has been reported in several ubiquitin-containing intraneuronal or intraglial inclusions such as neurofibrillary tangles, Pick bodies, Lewy bodies and glial cytoplasmic inclusions in various neurodegenerative diseases [7, 9, 27]. In the present study, we examined the immunoreactivity of p62 in ubiquitin-immunoreactive intraneuronal inclusions in five ALSD cases with a broad clinicopathological spectrum to investigate the contribution of p62 to the formation of these inclusions.
Patients and methods Patients The subjects comprised three males and two females with sporadic ALSD. The clinical summaries of these five cases are shown in
360 Table 1 Clinical summaries of the five ALSD cases (ALSD amyotrophic lateral sclerosis with dementia) Case
1
2
3
4
5
Sex Age at onset (years) Duration of illness (months) Interval from motor symptoms to dementia (months) Initial motor symptom Pyramidal signs Ophthalmoplegia Mechanical ventilation
Female 74 10 3 Upper limbs – – –
Female 61 36 12 Upper limbs + – –
Male 51 39 6 Upper limbs – – –
Male 45 60 –36a Upper limbs + – –
Male 40 137 >60b Bulbar + + (supranuclear) +
aDementia bNo
preceded motor symptoms dementia during “communicable” period
Table 1. The mean age (± SD) at onset was 54.2±12.1 years (range 40–74 years). The mean duration of illness was 56.4±43.3 months (range 10–137 months). The cases were numbered from 1 to 5 in order of duration (short to long) of illness. Initial motor symptoms were weakness of the upper limbs in four patients (cases 1, 2, 3 Fig. 1 Representative pathological findings in present cases. A A large-sized Bunina body (arrow) in a remaining anterior horn cell of the lumbar cord from case 5. B Medial portion of the rostral temporal cortex from case 2 showing mild neuronal loss with gliosis and neuropil microvacuolation in the II and III layers. C The substantia nigra from case 5 showing marked neuronal loss with gliosis without Lewy bodies. A–C Hematoxylin-eosin stain
and 4) and bulbar palsy in one patient (case 5). Dementia developed after the appearance of muscle weakness in three patients (cases 1, 2 and 3). In one patient (case 4), dementia and psychiatric symptoms appeared prior to the development of motor symptoms. One patient (case 5) survived 137 months under mechanical ventilation and developed ophthalmoplegia in the later stage, but not dementia or other psychiatric disorders during the “communicable” period. However, this case shows typical neuropathological findings of ALSD in extra-motor cortices and multiple system degeneration, as will be mentioned later.
361 Methods Neuropathological study Postmortem brains and spinal cords were fixed with 10% formalin and embedded in paraffin. Sections were stained with hematoxylin-eosin (HE) and by the Klüver-Barrera method. Selected sections were also stained with modified Bielschowsky, Holzer, phosphotungstic acid hematoxylin (PTAH) and periodic acidSchiff (PAS) methods as well as immunohistochemically with anti-ubiquitin, anti-tau and anti-α-synuclein antibodies. Immunohistochemistry for ubiquitin and p62 Adjacent 5-µm coronal sections from the temporal lobe including the hippocampus at the level of the lateral geniculate body were stained immunohistochemically using anti-ubiquitin and anti-p62 antibodies. After deparaffinization and rehydration, the sections were incubated in phosphate-buffered saline (PBS, pH 7.4) containing 3% hydrogen peroxide (H2O2) for 30 min to eliminate endogenous peroxidase activity. After three 5-min washes in PBS and blocking with normal serum, the sections were incubated with anti-ubiquitin (Dako, rabbit polyclonal, 1:1,000) or anti-p62/ZIP (C-terminal-specific, Santa Cruz Biotechnology, goat polyclonal, 1:1,000) antibodies overnight at 4°C. After three 5-min washes in Table 2 Distribution and severCase ity of degeneration in the five ALSD cases (+++ marked, Upper and lower motor neurons ++ moderate, + mild, – none) Hypoglossal nucleus Cervicothoracic cord Lumbosacral cord Bunina bodies Corticospinal tract
aOnly the caudal hippocampus was examined bIncluding caudate nucleus, putamen and nucleus accumbens septi
PBS, the sections were incubated in biotinylated secondary antibody for 1 h, and subsequently in horseradish-peroxidase-labeled streptavidin for 1 h. The sections were visualized with 0.02% 3,3’diaminobenzidine (DAB) in PBS containing 0.0005% H2O2. To enhance the signal, the sections for p62 immunohistochemistry were pretreated by microwave in 0.01 M citrate buffer (pH 6.0) at a high-power setting for 15 min. Counterstain was carried out with hematoxylin. Semiquantitative analysis In each case, the total number of intraneuronal inclusions immunoreactive for ubiquitin or p62 in the dentate gyrus was quantified in three series of adjacent sections.
Results Neuropathological findings In cases 1–4, loss of motor neurons and gliosis was more severe in the cervicothoracic cord and hypoglossal nucleus than in the lumbosacral cord. Degeneration of the 1
2
3
4
5
+++ +++ ++ Present +
+++ +++ ++ Present +
+++ +++ ++ Present +
+++ +++ ++ Present +
+++ +++ +++ Present +++
Cerebral cortices Frontal Rostral temporal Insular CA1–subiculum border Subcortical gliosis
+ + + + +
+ + + ++ +
+ + + –a +
+ + + ++ +
+ + + ++ +
Others Amygdala Basal nucleus of Meynert Neostriatumb Globus pallidus Subthalamic nucleus Thalamus Midbrain tegmentum Red nucleus Substantia nigra Locus coeruleus Pontine nuclei Dorsal vagal nucleus Inferior olivary nucleus
+ – – – – – – – ++ + – – –
– – – – + + – – +++ + – – –
+ – – ++ ++ ++ ++ ++ +++ + ++ – ++
++ – – – – – – – ++ – – – –
++ – – +++ +++ – ++ + +++ ++ ++ ++ +
Cerebellum Purkinje cell layer Granular layer Dentate nucleus Dorsal nucleus of Clarke Intermediolateral nucleus Nucleus of Onufrowicz Posterior column
+ – – – – – –
++ – + + – – –
+ – ++ – – – +
+ – – – – – –
+ + + +++ + – +
362
Fig. 2 Immunohistochemistry for ubiquitin (A, C) and p62 (B, D) in the granular layer of the dentate gyrus at the level of the lateral geniculate body. A Case 1, ubiquitin. B Case 1, p62. C Case 5, ubiquitin. D Case 5, p62. The proportion of p62-immunoreactive inclusions to ubiquitin-immunoreactive inclusions (p62/Ub ratio) in the granule cells was 6.3% in case 1 and 47.3% in case 5. Bars 50 µm
corticospinal tract was mild in these four cases. In case 5, the patient with the longest duration of illness, lower motor neurons were extensively affected, and degeneration of the corticospinal tract was severe. Bunina bodies in the remaining motor neurons were seen in all cases. Bunina bodies in these cases (Fig. 1A) tended to be larger than those in the cases of amyotrophic lateral sclerosis (ALS) without dementia. All cases showed mild neuronal loss with gliosis and neuropil microvacuolation in the II and III layers (Fig. 1B) of the frontal, rostral temporal and insular cortices and subcortical gliosis. Neuronal loss with gliosis at the border between CA1 and the subiculum was seen in four cases (cases 1, 2, 4 and 5). Degeneration of the amygdala was seen in four cases (cases 1, 3, 4 and 5). Some senile plaques and neurofibrillary tangles in the cerebral cortices were seen in case 1, but not in other cases. Neuronal loss in the substantia nigra without Lewy bodies (Fig. 1C) was seen in all cases. Degeneration of systems other than the motor neurons was more widespread and more severe in the cases with longer duration of illness. Especially in case 5, the patient with the longest duration of illness, the extrapyramidal and cerebellar systems were extensively affected. However, the neostriatum (caudate nucleus, putamen and nucleus accumbens septi) was well preserved even in case 5. Distribution and severity of degeneration in each case are shown in Table 2. Distribution of ubiquitin-immunoreactive intraneuronal inclusions Ubiquitin-immunoreactive (not argyrophilic, tau-negative, α-synuclein-negative) intracytoplasmic inclusions were
seen in the granule cells of the dentate gyrus and in the small neurons in the superficial layers of the entorhinal cortex in all cases. These inclusions were round, oval or crescent, and occasionally showed a circular pattern around the nucleus (Fig. 2). In some cases, such inclusions were also seen in the superficial layers of the frontal (in cases 1, 2 and 5), rostral temporal (in cases 1, 3, 4 and 5), cingulate (in case 5) or insular (in cases 1, 2, 3 and 5) cortices as well as in the amygdala (cases 1, 4 and 5). Similar inclusions were also seen in the medium-sized neurons in the neostriatum in four cases (not in case 4). There was no correlation between the number of inclusions and severity of dementia, duration of illness or neuropathological severity. Inclusions of this type were not recognized in the brain stem and spinal cord in any of the cases. Bunina bodies were not immunoreactive for ubiquitin in any case. Distribution and frequency of inclusions in each case is shown in Table 3. Proportion of p62-immunoreactive inclusions Some ubiquitin-immunoreactive intraneuronal inclusions in the dentate gyrus and entorhinal cortex were also p62 immunoreactive in all cases. However, the proportion of p62-immunoreactive inclusions to the total number of Table 3 Distribution and frequency of ubiquitin-immunoreactive intraneuronal inclusions (+++ numerous,++ moderate number, + a few, – none) Case
1
2
3
4
5
Dentate gyrus Entorhinal cortex Amygdala Frontal cortex Rostral temporal cortex Cingulate cortex Insular cortex Neostriatuma
+++ + + + + – + +
+++ + – + – – + +
++ + – – + – + +
+++ + + – + – – –
+++ + + + + + + +
aIncluding
caudate nucleus, putamen and nucleus accumbens septi
363 Table 4 Number of ubiquitin- and p62-immunoreactive inclusions in the granule cells of the dentate gyrus at the level of the lateral geniculate body (Ub ubiquitin-immunoreactive inclusions, p62 p62-immunoreactive inclusions) Case
1
2
3
4
5
Ub (number/ 5-µm section) P62 (number/ 5-µm section) p62/Ub ratio (%)
436.7
244.7
120.7
564.3
542.7
27.3
110.3
31.7
71.0
256.7
6.3
45.1
26.3
12.6
47.3
ubiquitin-immunoreactive inclusions (p62/Ub ratio) in the dentate gyrus was varied among all cases (Table 4, Fig. 2). The mean p62/Ub ratio was 27.5±16.6% (range 6.3– 47.3%). There was no correlation between p62/Ub ratio and severity of dementia, duration of illness or neuropathological severity.
Discussion Neuropathological studies of ALSD have revealed mild neuronal loss with gliosis and neuropil microvacuolation in the II and III layers of the extra-motor cortices in addition to degeneration of upper and lower motor neurons, common findings in ALS without dementia [2, 5, 6, 10, 11, 13, 14, 15, 25, 26]. Furthermore, degeneration of other systems, especially neuronal loss without Lewy bodies in the substantia nigra, has been shown in most of ALSD cases. However, the extent and severity of degeneration in other regions varies among the reported cases [2, 5, 6, 10, 11, 15, 26]. In our present cases, degeneration of systems other than the motor neurons was more widespread and more severe in the cases with longer duration of illness. In ALS without dementia, some cases with long duration of illness under mechanical ventilation show degeneration of multiple systems [4, 12, 19, 21], while degeneration in other cases is quite restricted to the upper and lower motor neurons even after more than 10 years of clinical course [4]. On the other hand, all cases with ALSD may be destined to develop degeneration of multiple systems. Ubiquitin-immunoreactive, tau-negative intraneuronal inclusions in ALSD were first reported by Okamoto et al. in 1991 [16]. However, the main constituent of these inclusions is still unknown. These inclusions have also been shown in several cases without obvious dementia or without pathological changes in the frontotemporal neocortical area [1, 16, 24, 25]. This study showed that these inclusions were seen both in the neuropathologically affected areas (e.g., frontal and rostral temporal cortices, amygdala) and non-affected areas (e.g., dentate gyrus, neostriatum). Especially, granule cells in the dentate gyrus were well preserved, in spite of the presence of the most numerous inclusions. These findings suggest that the formation of inclusions does not directly cause neuronal death. We show here immunoreactivity of p62 in ubiquitinimmunoreactive inclusions in the granule cells of the dentate gyrus in ALSD. Shin [20] and Vadlamudi et al. [23]
have reported that p62, a non-proteasomal protein, previously identified as a phosphotyrosine-independent ligand for the p56lck SH2 domain, also binds multiubiquitin chains and forms a cytoplasmic structure “sequestosome”, which serves as a storage place for ubiquitinated proteins. Kuusisto et al. [7, 9] have reported the immunoreactivity of p62 in several ubiquitin-containing intraneuronal or intraglial inclusions in tauopathies (neurofibrillary tangles, Pick bodies) and synucleinopathies (Lewy bodies, glial cytoplasmic inclusions), suggesting the presence of a common mechanism in the aggregation of ubiquitinated proteins via p62 in both tauopathies and synucleinopathies. They hypothesized that p62 was noncovalently bound to ubiquitin via its C-terminal domain, and dimerized via its N-terminal areas, consequently forming aggregates of ubiquitinated proteins [7]. The same mechanism may contribute to the formation of ubiquitin-immunoreactive inclusions in ALSD. In addition, they have reported the induction of p62 expression during experimental apoptosis and proteasomal inhibition in cultured neuronal cells, suggesting a protective mechanism in neuronal degeneration [8]. Furthermore, Samuels et al. [18] have reported that transfection of an antisense p62 construct into PC12 cells diminished neurite outgrowth induced by nerve growth factor. Although our results suggest that p62 contributes to the aggregation of the main constituent of ubiquitin-immunoreactive inclusions in ALSD, the formation of the inclusions may produce a non-toxic but rather protective effect. It may be for this reason that the number of inclusions and p62/Ub ratio do not correlate with the severity of dementia, duration of illness or neuropathological severity. In conclusion, our present study demonstrates that ubiquitin-binding protein p62 is a constituent of ubiquitinimmunoreactive intraneuronal inclusions, one of the characteristic findings of ALSD, suggesting that p62 contributes to the inclusion formation that may protect the neurons from degeneration. Further studies, especially the determination of the main constituent of the inclusions will be necessary to elucidate the significance of these inclusions in ALSD.
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