Immunohistochemical visualization of afferent nerve terminals in human globus pallidus and its alteration in neostriatal neurodegenerative disorders

Summary The afferent nerve terminal in the human globus pallidus, which receives the projection nerve fibers from both the striatum and the subthalamic nucleus, were clearly visualized immunohistochemically using antibodies to calcineurin, synaptophysin, Met-enkephalin (MEnk) and substance P (SP). In normal control case, MEnk and SP-like immunoreactivities were densely localized in the external and internal pallidal segments, respectively, whereas calcineurin and synaptophysin were distributed throughout the globus pallidus. Calcineurin, synaptophysin, MEnk and SP-like immunoreactive peroxidase products decorated most of the long radiating dendrites and the cell bodies of the pallidal neurons. In the cases with Huntington's disease (HD) and striatonigral degeneration (SND), marked loss of calcineurin, MEnk and SP-like immunoreactivities was seen in the globus pallidus corresponding to areas of striatal neurodegeneration, whereas synaptophysin immunoreactivity remained in areas which revealed almost complete loss of calcineurin, MEnk and SP-like immunoreactivities. Calcineurin, MEnk and SP, which show difference in their localization patterns, may provide reliable markers for the striatal efferent nerve terminals, and synaptophysin for the entire pallidal afferent nerve terminals. This report demonstrates the distribution patterns of these neurochemical molecules in the globus pallidus with HD and SND.     

Numerous FUS‐positive inclusions in an elderly woman with motor neuron disease

We report an autopsy case of a 75‐year‐old Japanese woman with motor neuron disease (MND) showing numerous neuronal and glial inclusions immunostained with anti‐fused in sarcoma (FUS) antibody. At 73 years, she received a diagnosis of MND and died of respiratory insufficiency 2 years later. No mutation was found in all exons of the FUS gene. Neuropathological examination revealed a reduced number of anterior horn cells and degeneration of the pyramidal tracts. Neither Bunina bodies nor inclusions positive for ubiquitin/phosphorylated TAR DNA binding protein of 43 kD (pTDP‐43), such as skein‐like or round inclusions, were observed. However, basophilic inclusions (BIs) were frequently observed in the remaining neurons of the anterior horns, facial nuclei, hypoglossal nuclei, vestibular nuclei, dentate nuclei and inferior olivary nuclei. In an immunohistochemical analysis, the BIs showed strong immunoreactivity with anti‐FUS and anti‐ubiquitin‐binding protein p62 (p62) antibodies. The nuclear staining of FUS was preserved in some neurons with FUS‐positive inclusions, and a few FUS‐positive glial inclusions were found. FUS‐positive inclusions were more common than p62‐positive inclusions in some anatomical regions, and in some neurons, p62 immunoreactivity was observed in only parts of the BIs. These results suggest that BI formation and TDP‐43 aggregation have different pathogenic mechanisms, and FUS may play an important role in the pathogenesis of MND with BIs. This patient has the oldest reported age of onset for MND with BIs, and clinical features observed in this patient were indistinguishable from those of classic sporadic MND. Therefore, we consider that the age of onset and clinical features of FUS‐related disorders may be variable.     

An autopsied case of sporadic adult‐onset amyotrophic lateral sclerosis with FUS‐positive basophilic inclusions

Basophilic inclusions (BIs), which are characterized by their staining properties of being weakly argyrophilic, reactive with Nissl staining, and immunohistochemically negative for tau and transactive response (TAR) DNA‐binding protein 43 (TDP‐43), have been identified in patients with juvenile‐onset amyotrophic lateral sclerosis (ALS) and adult‐onset atypical ALS with ophthalmoplegia, autonomic dysfunction, cerebellar ataxia, or a frontal lobe syndrome. Mutations in the fused in sarcoma gene (FUS) have been reported in cases of familial and sporadic ALS, and FUS immunoreactivity has been demonstrated in basophilic inclusion body disease (BIBD), neuronal intermediate filament inclusion disease (NIFID), and atypical frontotemporal lobar degeneration with ubiquitin‐positive and tau‐negative inclusions (aFTLD‐U). In the present study, we immunohistochemically and ultrastructurally studied an autopsy case of sporadic adult‐onset ALS with numerous BIs. The patient presented with the classical clinical course of ALS since 75 years of age and died at age 79. Postmortem examination revealed that both Betz cells in the motor cortex and motor neurons in the spinal cord were affected. The substantia nigra was spared. Notably, BIs were frequently observed in the motor neurons of the anterior horns, the inferior olivary nuclei, and the basal nuclei of Meynert. BIs were immunopositive for p62, LC3, and FUS, but immunonegative for tau, TDP‐43, and neurofilament. Ultrastructurally, BIs consisted of filamentous or granular structures associated with degenerated organelles with no limiting membrane. There were no Bunina bodies, skein‐like inclusions, or Lewy‐like inclusions. All exons and exon/intron boundaries of the FUS gene were sequenced but no mutations were identified.     

Striosomal arrangement of met-enkephalin and substance P expression in parkinsonism-dementia complex on Guam

Summary This report concerns a topographic immunohistochemcal analysis of Met-enkephalin (MEnk) and substance P (SP) expression in the striatum from seven patients with parkinsonism-dementia complex on Guam (PDC). The striatum of seven neurologically normal subjects served as controls. MEnk-positive striosomes were readily visualized in both the caudate nucleus and the putament of the PDC cases. Although the immunoreactivity was less than that of MEnk, SP-positive patches were detected in the PDC striatum. The patches were similar to those seen in the normal controls. These results provide additional evidence that the striatum and its efferent system appear to be preserved in patients with PDC.Supported in part by grants from the Amyotrophic Lateral Sclerosis Association and Sumitomo Pharmaceuticals Co. Ltd.     

Phenotypic variation in ALS]

Making a diagnosis of typical amyotrophic lateral sclerosis (ALS) is not a tough job, but when it comes to atypical forms of motor neuron disease (MND) which are not uncommon in clinical setting, we may have some difficulty to diagnose ALS/MND. There is striking phenotypic variation in sporadic ALS/MND, such as frail arm syndrome (brachial amyotrophic diplegia), pseudopolyneuritic form, hemiplegic type, ALS/MND with markedly extended involvement beyond the motor system, and MND with basophilic inclusion bodies. These variations must be recognized if physicians are to tailor advice on disease progression, prognosis, drug therapy, and care to the needs of the individual. Clinical trials of new therapeutic agents have been performed, on the assumption that patients with ALS/MND have the same underlying etiology, addressing the heterogeneous population of the patients under a single diagnostic category. This can be detrimental to the well-being of the individual, because clinical heterogeneity may mask drug effects in clinical trials. The attempt to categorize subgroups based on the clinical and pathological backgrounds within the spectrum of ALS/MND may be a critical step in facilitating clinical research in ALS/MND. Definition of clinicopathologic syndromes in patients with ALS/MND is an important challenging task that cannot be ignored.     

Inversion of T/TMP ratio in ALS: a specific finding?

Thiamine (T) and thiamine monophosphate (TMP) levels were determined by an electrophoretic fluorometric method in the CSF of patients with typical sporadic ALS (50 cases), in other motor neuron diseases (MND) (14 cases) and in patients with upper and/or lower motor neuron lesions of varying origin (disseminated sclerosis, polyneuropathy, spondylotic myelopathy). T/TMP ratio was ≥1 in a high percentage of patients with typical sporadic ALS (94%), in 35.7% of cases with other MND, while it was below 1 in the all other patients. The decrease of TMP with the inversion of the T/TMP ratio is a finding highly specific to typical sporadic ALS. This text was presented as communication at the Italo-Polish meeting held in Rome an 20–21 April 1985 arranged by the Società Italiana di Neurologia     

Met-enkephalin immunoreactivity in the basal ganglia in Parkinson's disease and striatonigral degeneration

This study concerns the expression of Met-enkephalin (MEnk) in the striatum and the external segment of the globus pallidus proper (GPe) in normal controls, idiopathic Parkinson's disease (PD), and striatonigral degeneration (SND). For this purpose, we developed a sensitive immunoperoxidase technique to visualize MEnk-positive patches in routinely prepared formalin-fixed paraffin-embedded striatal tissues. In comparison with normal controls, MEnk-positive patches and pallidal MEnk-positive axon terminals were strongly present in patients with PD, showing characteristic distribution patterns. By comparison, in SND patients, there was striking diminution of MEnk staining in the putamen and ventrolateral portion of the GPe, while MEnk patches were persistent in the caudate nucleus.     

FUS/TLS-immunoreactive neuronal and glial cell inclusions increase with disease duration in familial amyotrophic lateral sclerosis with an R521C FUS/TLS mutation

Basophilic inclusions (BIs) are pathological features of a subset of frontotemporal lobar degeneration disorders, including sporadic amyotrophic lateral sclerosis (ALS) and familial ALS (FALS). Mutations in the fused in sarcoma/translocated in liposarcoma (FUS/TLS) gene have recently been identified as a cause of FALS. The FUS/TLS-immunoreactive inclusions are consistently found in cases of frontotemporal lobar degeneration with BIs; however, the association between ALS cases with BIs and FUS/TLS accumulation is not well understood. We used immunohistochemistry to analyze 3 autopsy cases of FALS with the FUS/TLS mutation and with BIs using anti-FUS/TLS antibodies. The disease durations were 1, 3, and 9 years. As the disease duration becomes longer, there were broader distributions of neuronal and glial FUS/TLS-immunoreactive inclusions. As early as 1 year after the onset, BIs, neuronal cytoplasmic inclusions and glial cytoplasmic inclusions were found in the substantia nigra in addition to the anterior horn of the spinal cord. Glial cytoplasmic inclusions are found earlier and in a wider distribution than neuronal cytoplasmic inclusions. The distribution of FUS/TLS-immunoreactive inclusions in FUS/TLS-mutated FALS with BIs was broader than that of BIs alone, suggesting that the pathogenetic mechanism may have originated from the FUS/TLS proteinopathy.     

The Golgi apparatus is fragmented in spinal cord motor neurons of amyotrophic lateral sclerosis with basophilic inclusions

The mechanisms of neuronal death in amyotrophic lateral sclerosis (ALS) are not known. A pathological aggregation of cytoplasmic constituents in the form of variety of inclusions may play a role in the pathogenesis of neuronal death. Cytoplasmic basophilic inclusions (BIs) in motor neurons are commonly found in sporadic juvenile ALS. The functional significance of these inclusions is not known, i.e., whether they represent a protective reaction for the isolation of abnormal products from the cytoplasm, or a sign of irreversible neuronal damage. To gain insights on the significance of BIs we asked whether neurons with BIs had an intact or fragmented Golgi apparatus (GA), a sign of neuronal degeneration reported not only in sporadic and familial ALS with mutations of the Cu/Zn superoxide dismutase gene (SOD1), but also in transgenic mice expressing the G93A mutation of SOD1. In these mice fragmentation of the GA of spinal cord motor neurons was found months before the onset of paralysis. We report here that all neurons bearing the inclusions showed fragmentation and reduced number of GA. These results suggest that common pathogenetic mechanisms are involved in the production of BIs and in the fragmentation of the GA.     

Dopaminergic deficit in amyotrophic lateral sclerosis assessed with [I-123] IPT single photon emission computed tomography

Dopamine transporter imaging was performed in 18 patientswith sporadic amyotrophic lateral sclerosis (ALS) and 11 age matched controls with [I-123] IPT(N-(3-iodopropen-2-yl)-2β-carbomethoxy-3β(4-chlorophenyl)-tropane), a new cocaine analogue that selectively binds to the dopamine transporter located on dopaminergic nerve terminals. Image analysis showed that striatal IPT binding was moderately but significantly reduced in the ALS group compared with controls (p<0.01). The reduction of IPT binding was similar for patients with bulbar onsetcompared with those with limb onset. There was no correlation betweenvalues for uptake of striatal IPT and the age of the patients or theduration of the disease. These data indicate that nigrostriatal dopaminergic neurons are subclinically affected in a subset of patientswith sporadic ALS.

     

An MND/ALS phenotype associated with C9orf72 repeat expansion: Abundant p62‐positive,TDP‐43‐negative inclusions in cerebral cortex,hippocampus and cerebellum but without associated cognitive decline

The transactive response DNA binding protein (TDP‐43) proteinopathies describe a clinico‐pathological spectrum of multi‐system neurodegeneration that spans motor neuron disease/amyotrophic lateral sclerosis (MND/ALS) and frontotemporal lobar degeneration (FTLD). We have identified four male patients who presented with the clinical features of a pure MND/ALS phenotype (without dementia) but who had distinctive cortical and cerebellar pathology that was different from other TDP‐43 proteinopathies. All patients initially presented with weakness of limbs and respiratory muscles and had a family history of MND/ALS. None had clinically identified cognitive decline or dementia during life and they died between 11 and 32 months after symptom onset. Neuropathological investigation revealed lower motor neuron involvement with TDP‐43‐positive inclusions typical of MND/ALS. In contrast, the cerebral pathology was atypical, with abundant star‐shaped p62‐immunoreactive neuronal cytoplasmic inclusions in the cerebral cortex, basal ganglia and hippocampus, while TDP‐43‐positive inclusions were sparse. This pattern was also seen in the cerebellum where p62‐positive, TDP‐43‐negative inclusions were frequent in granular cells. Western blots of cortical lysates, in contrast to those of sporadic MND/ALS and FTLD‐TDP, showed high p62 levels and low TDP‐43 levels with no high molecular weight smearing. MND/ALS‐associated SOD1, FUS and TARDBP gene mutations were excluded; however, further investigations revealed that all four of the cases did show a repeat expansion of C9orf72, the recently reported cause of chromosome 9‐linked MND/ALS and FTLD. We conclude that these chromosome 9‐linked MND/ALS cases represent a pathological sub‐group with abundant p62 pathology in the cerebral cortex, hippocampus and cerebellum but with no significant associated cognitive decline.     

German-Canadian family (family A) with parkinsonism, amyotrophy, and dementia - Longitudinal observations

Etiology of Parkinson's disease (PD), amyotrophy lateral sclerosis (ALS), and Alzheimer's disease (AD) remains uncertain. Environmental factors probably play a role, but genetic influences may predispose certain individuals to develop each of these major neurodegenerative disorders. We describe our longitudinal observations concerning a Canadian family traced to Northern Germany. Autosornal dominant inheritance has been established. Affected members present with L-dopa responsive parkinsonism and amyotrophy. In the German portion of the family some individuals displayed only dementia or focal dystonia. Linkage analysis studies performed with polymorphic markers associated with 13 candidate genes provided no significant evidence for linkage with any of the genes examined. Positron emission tomography with [(18)F]-6-fluoro-L-dopa (FD) and [su11C]-raclopride (raclopride) of one affected subject revealed reduced striatal FD uptake particularly in putamen, and an increased raclopride striatum/background ratio. Postmortem levels of dopamine and its metabolites were greatly reduced in caudate and putamen of two patients. There was substantial neuronal loss in the substantia nigra and the presence of abundant eosinophilic granules (different than Lewy bodies) in surviving neurons. One of them also showed mild loss of anterior horn cells, while another showed abundant senile plaques and some neurofibrillary tangles in distribution and intensity typical of mild to moderate AD. Our report further describes this unique family with a combination of clinical features of PD, ALS, and AD. By studying kindreds like this we may learn more about the pathophysiology of sporadic forms of PD, ALS, or even AD.     

Inhomogeneity of the putaminal lesion in striatonigral degeneration

Summary An immunohistochemical topographic study was carried out in the putamen from three patients with striatonigral degeneration (SND) using antibody to calcineurin (CaN), a neurochemical marker for the striatal medium-size spinous neurons. In patients with SND, there was significant depletion of CaN immunoreactivity in the putamen with the caudal and lateral portion of the putamen being consistently and severely affected. In addition, the SND patients showed an inhomogeneous distribution pattern of residual CaN staining in the putamen, where remaining CaN immunoreactivity appeared as a characteristic patchwork of islands resembling the striosomes observed by the tyrosine hydroxylase or Met-enkephalin immunostaining in the putamen from normal individuals. This finding may account for the fact that there are subregional and compartmental differences in susceptibility of the medium-sized spinous neurons in the putamen with SND.     

Increased cerebrospinal fluid levels of substance P in patients with amyotrophic lateral sclerosis

To clarify the mechanism of brain and spinal cord impairment in amyotrophic lateral sclerosis (ALS), we measured the cerebrospinal fluid (CSF) levels of substance P (SP) in 11 patients with sporadic ALS. Findings were compared with those obtained in controls and diseased controls. The CSF SP levels of patients with ALS, and particularly in patients with a disease duration of less than 2.5 years, were significantly higher than those in controls. These findings strongly suggested that SP may play an important role in the pathophysiology of ALS.     

Neutral endopeptidase-24.11, μ and δ opioid receptors after selective brain lesions: an autoradiographic study

The cellular localization of the rat brain neutral endopeptidase (NEP, EC 3.4.24.11) was investigated by quantitative autoradiography of the enzyme inhibitor [3H]N-[(2RS)-3-hydroxyaminocarbonyl-2-benzyl-1-oxopropyl]glycine ([3H]HACBO-Gly) after lesions of the striatum, nigrostriatal and corticostriatal pathways. The effect of these lesions on NEP levels was compared with that on delta and mu opioid receptors, selectively labeled with [3H]Tyr-D-Thr-Gly-Leu-Thr ([3H]DTLET) and [3H]Tyr-D-Ala-Gly-MePhe-Glycinol ([3H]DAGO), respectively. Twenty-one days after injection of kainate in the caudate putamen (CP), the NEP level was locally decreased (52%) but the time course of this decrease was different from that of mu and delta opioid receptors: [3H]DAGO binding was diminished by 40% from day 2 whereas that of [3H]DTLET was reduced by 51% from day 7. Kainic acid injection in the CP induced in the globus pallidus (GP) and substantia nigra (SN) a distant reduction of the 3 opioid markers. Likewise after injection of colchicine in the CP, [3H]HACBO-Gly binding was decreased in the GP (60%) and SN (58%), [3H]DTLET binding was reduced by 54 and 55% in the GP and SN, respectively and [3H]DAGO labeling was diminished by 49% in the GP, and 58% in the SN. Finally, lesion of the nigrostriatal dopamine pathway by 6-hydroxydopamine did not induce any change of NEP level in the CP and GP whereas delta and mu opioid receptor levels were diminished respectively by 25 and 29% in the CP, and 45 and 39% in the GP, a new finding of the present study. Taken together these data suggest that NEP is in part associated with striatal intrinsic neurons. In the GP and SN, a large part of NEP seems to be presynaptically associated with nerve terminals endowed with mu and delta opioid receptors, which originate from efferent striatal neurons. In contrast to opioid receptors in the CP, the NEP appears not to be associated with dopaminergic nerve terminals originating from the SN. Cortical ablation did not affect any of the opioid markers.     

Distribution of neuropeptide Y immunoreactivity in the basal forebrain and upper brainstem of the squirrel monkey (Saimiri sciureus)

The distribution of neuropeptide Y (NPY) immunoreactivity in the brain of the squirrel monkey (Saimiri sciureus) was studied by means of the indirect immunofluorescence, peroxidase-antiperoxidase, and avidin-biotin-complex methods. The antiserum used was raised in rabbits and did not show any significant crossreactivity with related peptides including peptide YY and avian pancreatic polypeptide. In the upper brainstem of the squirrel monkey a dense NPY-immunoreactive terminal field is seen in lateral parabrachial area, locus coeruleus, and interpeduncular nucleus. A small group of NPY-immunoreactive cell bodies is present in the lateral habenula and a moderate number of NPY-immunoreactive fibers occurs in periaqueductal gray and nucleus raphe pallidus. The substantia nigra (SN) appears mostly devoid of NPY immunoreactivity whereas the ventral tegmental area contains a few reactive fibers. In the hypothalamus the medial preoptic area as well as the arcuate and paraventricular nuclei receive a strikingly dense NPY innervation. In addition, numerous NPY-positive cell bodies are found within the dorsomedial half of the supraoptic nucleus but very few are seen in paraventricular nucleus. A large number of NPY-immunoreactive cell bodies is also present in arcuate nucleus. In the basal telencephalon NPY-immunoreactive cells abound mostly in striatum, but some are also found in the amygdala (particularly basal, central, and lateral amygdaloid nuclei), the claustrum, and in the bed nucleus of the stria terminalis. Intensely reactive network of NPY-immunoreactive fibers is also present in all of these structures. In striatum, the numerous, fine and non-varicose NPY-immunoreactive fibers, as well as the NPY-positive cell bodies, are slightly more abundant in caudate nucleus than in putamen. The globus pallidus (GP) is mostly devoid of NPY-immunoreactive fibers and terminals. The fact that the two major recipient structures of striatal outflow (SN and GP) do not receive significant NPY input suggests that the striatal NPY-containing neurons are intrinsically organized.     

Striatal c-fos Induction by Cocaine or Apomorphine Occurs Preferentially in Output Neurons Projecting to the Substantia Nigra in the Rat

Fluorogold or rhodamine-labelled latex beads were injected in the substantia nigra (SN) or the globus pallidus (GP) in order retrogradely to label striatal output neurons that project to the two target structures. Ten days later, striatal c-fos was induced by systemic administration of cocaine (five normal rats; 25 mg/kg cocaine i.p. 2 h before killing) or apomorphine (five unilaterally dopamine-denervated rats; 0.25 mg/kg apomorphine s. c. 2 h before killing), and detection of the Fos protein in the striatum was achieved by immunofluorescence. Sections through the caudate-putamen that displayed good labelling from both SN and GP were selected for a quantitative analysis: the number of retrogradely labelled cells that exhibited Fos immunoreactivity, as well as the total number of retrogradely labelled cells located within a grid (0.16 mm² in size) were counted manually at 25 x magnification. Cocaine induced a proportionally higher c-fos expression in striate-nigral compared to striate-pallidal neurons, whereas apomorphine activated Fos almost exclusively in striate-nigral neurons. The present findings are consistent with the idea that striatal c-fos induction by dopaminergic agents is primarily mediated by an interaction with D1 -receptors, which are thought to be selectively localized on neurons projecting to SN.     

Reduced expression of BTBD10 in anterior horn cells with Golgi fragmentation and pTDP‐43‐positive inclusions in patients with sporadic amyotrophic lateral sclerosis

Overexpression of BTBD10 (BTB/POZ domain‐containing protein 10) suppresses G93A‐superoxide dismutase 1 (SOD1)‐induced motor neuron death in a cell‐based amyotrophic lateral sclerosis (ALS) model. In the present study, paraffin sections of spinal cords from 13 patients with sporadic ALS and 10 with non‐ALS disorders were immunostained using a polyclonal anti‐BTBD10 antibody. Reduced BTBD10 expression in the anterior horn cells was more frequent in spinal cords from ALS patients than in cords from patients with non‐ALS disorders. We further investigated the relationship between the level of BTBD10 immunoreactivity and the morphology of the Golgi apparatus (GA) and the presence of phosphorylated TAR‐DNA‐binding protein 43 (pTDP‐43). Mirror sections of spinal cords from five sporadic ALS cases were immunostained with antibodies against BTBD10 and trans‐Golgi‐network (TGN)‐46 or pTDP‐43. Whereas 89.7–96.5% of the neurons with normal BTBD10 immunoreactivity showed normal GA morphology and no pTDP‐43 cytoplasmic aggregates, 86.2–94.3% of the neurons with reduced BTBD10 expression showed GA fragmentation and abnormal pTDP‐43 aggregates. These findings suggest that reduced BTBD10 expression is closely linked to the pathogenesis of sporadic ALS.