Inclusions In Amyotrophic Lateral Sclerosis Research Articles

Frontotemporal Lobar Degeneration: Current Concepts in the Light of Recent Advances

Work done over the past decade has led to a molecular understanding of frontotemporal lobar degeneration (FTLD), a deadly disease that afflicts patients in mid-life. It is a common cause of dementia, second only to Alzheimer's disease in the population below 65 years of age. Neuroanatomical and neurobiological substrates have been identified for the three major subtypes of FTLD and these discoveries have broadened the FTLD spectrum to include amyotrophic lateral sclerosis (ALS). Mutations in MAPT were found to cause frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), a familial disorder with filamentous tau inclusions in nerve cells and glial cells. FTDP-17 can result in clinical syndromes that closely resemble progressive supranuclear palsy, corticobasal degeneration and Pick's disease. More recently, mutations in three genes (VCP, CHMP2B and PGRN) have been found to cause FTLD with ubiquitin-positive, tau-negative neuronal inclusions (FTLD-U). They explain a large proportion of inherited FTLD-U. It remains to be seen whether dementia lacking distinctive histopathology (DLDH) constitutes a third disease category, as many of these cases are now being reclassified as FTLD-U. Recently, TAR DNA-binding protein-43 (TDP-43) has been identified as a key protein of the ubiquitin inclusions of FTLD-U and ALS. Thus, for familial forms of FTLD and related disorders, we now know the primary etiologies and accumulating proteins. These findings are pivotal for dissecting the pathways by which different etiologies lead to the varied clinicopathological presentations of FTLD.

LRRK2 Expression in Normal and Pathologic Human Brain and in Human Cell Lines

Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) have been recently identified in families with autosomal-dominant late-onset Parkinson disease. We report that by reverse transcriptase-polymerase chain reaction, the mRNA of LRRK2 is expressed in soluble extracts of human brain, liver, and heart and in cultured human astrocytes, microglia, and oligodendroglia as well as in human neuroblastoma cell lines. We find by Western blotting using a polyclonal antibody of the leucine-rich repeat kinase 2 protein (Lrrk2) specific for C-terminal residues 2511-2527 that an apparent full-length protein and several of its fractions are expressed in soluble extracts of normal human brain. By immunocytochemistry, the antibody recognizes neurons, and more weakly astrocytes and microglia, in normal brain tissue. It intensely labels Lewy bodies in Parkinson disease and related neurodegenerative disorders. It also labels a subset of neurofibrillary tangles in Alzheimer disease and the Parkinsonism dementia complex of Guam (PDCG). It labels thorn-shaped astrocytes and oligodendroglial coiled bodies in PDCG; oligodendroglial inclusions in multiple system atrophy; Pick bodies in Pick disease; nuclear and cytoplasmic inclusions in Huntington disease; and intraneuronal and glial inclusions in amyotrophic lateral sclerosis. In summary, LRRK2 is constitutively expressed in neurons and also in glial cells of human brain. It strongly associates with pathological inclusions in several neurodegenerative disorders.

Defective axonal transport of neurofilament proteins in neurons overexpressing peripherin

Peripherin is a type III neuronal intermediate filament detected in motor neuron inclusions of amyotrophic lateral sclerosis (ALS) patients. We previously reported that overexpression of peripherin provokes late-onset motor neuron dysfunction in transgenic mice. Here, we show that peripherin overexpression slows down axonal transport of neurofilament (NF) proteins, and that the transport defect precedes by several months the appearance of axonal spheroids in adult mice. Defective NF transport by peripherin up-regulation was further confirmed with dorsal root ganglia (DRG) neurons cultured from peripherin transgenic embryos. Immunofluorescence microscopy and western blotting revealed that excess peripherin provokes reduction in levels of hyperphosphorylated NF-H species in DRG neurites. Similarly the transport of a green fluorescent protein (GFP)-tagged NF-M, delivered by means of a lentiviral construct, was impaired in DRG neurites overexpressing peripherin. These results demonstrate that peripherin overexpression can cause defective transport of type IV NF proteins, a phenomenon that may account for the progressive formation of ALS-like spheroids in axons.

Monomeric Cu,Zn-superoxide Dismutase Is a Common Misfolding Intermediate in the Oxidation Models of Sporadic and Familial Amyotrophic Lateral Sclerosis

Proteinacious intracellular aggregates in motor neurons are a key feature of both sporadic and familial amyotrophic lateral sclerosis (ALS). These inclusion bodies are often immunoreactive for Cu,Zn-superoxide dismutase (SOD1) and are implicated in the pathology of ALS. On the basis of this and a similar clinical presentation of symptoms in the familial (fALS) and sporadic forms of ALS, we sought to investigate the possibility that there exists a common disease-related aggregation pathway for fALS-associated mutant SODs and wild type SOD1. We have previously shown that oxidation of fALS-associated mutant SODs produces aggregates that have the same morphological, structural, and tinctorial features as those found in SOD1 inclusion bodies in ALS. Here, we show that oxidative damage of wild type SOD at physiological concentrations ( approximately 40 microm) results in destabilization and aggregation in vitro. Oxidation of either mutant or wild type SOD1 causes the enzyme to dissociate to monomers prior to aggregation. Only small changes in secondary and tertiary structure are associated with monomer formation. These results indicate a common aggregation prone monomeric intermediate for wild type and fALS-associated mutant SODs and provides a link between sporadic and familial ALS.

Expression of peripherin in ubiquinated inclusions of amyotrophic lateral sclerosis

We evaluated the expression of the type III intermediate filament (IF) protein, peripherin (PRP), in ubiquinated inclusions of motor neurons in amyotrophic lateral sclerosis (ALS). A previous study showed that overexpression of PRP in transgenic mice induces motor neuron disease with formation of PRP-containing inclusions before onset of symptoms [J. Cell Biol. 147 (3) (1999) 531]. To determine whether PRP inclusions occur in the human disease, we applied doublelabeling immunofluorescence to paraffin sections of the spinal cord obtained by autopsy of 40 ALS patients with sporadic disease and 39 controls. Inclusions that expressed immunoreactive ubiquitin and peripherin were recorded by video camera, and the sections were stained by hematoxylin and eosin (H&E) to define morphology. Lewy body-like inclusions (LBLIs) were seen in motor neuron perikarya of 9 of 40 ALS cases and none in controls; all LBLIs expressed peripherin. Skein-like inclusions (SLIs) were identified by ubiquitin, but did not express PRP with rare exceptions. Neither skein-like inclusions nor LBLIs expressed αB-crystallin, neurofilament protein (NF-L, NF-M and NF-H subunits), α-internexin, actin or α-synuclein. Immunoblot of the whole spinal cord exhibited a single 57-kDa band of peripherin in ALS patients and controls. Our data document the expression of peripherin in LBLIs, which may provide a clue to the pathogenesis of neurodegeneration in ALS.

Dorfin Localizes to the Ubiquitylated Inclusions in Parkinson's Disease, Dementia with Lewy Bodies, Multiple System Atrophy, and Amyotrophic Lateral Sclerosis

In many neurodegenerative diseases, the cytopathological hallmark is the presence of ubiquitylated inclusions consisting of insoluble protein aggregates. Lewy bodies in Parkinson's disease and dementia with Lewy bodies disease, glial cell inclusions in multiple system atrophy, and hyaline inclusions in amyotrophic lateral sclerosis (ALS) are representative of these inclusions. The elucidation of the components of these inclusions and the mechanisms underlying inclusion formation is important in uncovering the pathogenesis of these disorders. We hypothesized that Dorfin, a perinuclearly located E3 ubiquitin ligase, participates in the formation of ubiquitylated inclusions in a wide range of neurodegenerative diseases. Here, we report that affinity-purified anti-Dorfin antibody labeled ubiquitylated inclusions of Parkinson's disease, dementia with Lewy bodies disease, multiple system atrophy, and sporadic and familial ALS. A double-immunofluorescence study revealed that Dorfin shows a distribution pattern parallel to that of ubiquitin. Furthermore, by a filter trap assay, we detected that Dorfin is present in the ubiquitylated high-molecular weight structures derived from these diseases. These results suggest that Dorfin plays a crucial role in the formation of ubiquitylated inclusions of alpha-synucleinopathy and ALS. However, because we failed to show the direct binding of alpha-synuclein with Dorfin, future investigations into the binding partner(s) of Dorfin will be needed to deepen our understanding of the pathophysiology of alpha-synucleinopathy and ALS.

Cognitive dysfunction in patients with amyotrophic lateral sclerosis is associated with spherical or crescent-shaped ubiquitinated intraneuronal inclusions in the parahippocampal gyrus and amygdala, but not in the neostriatum.

Skeins or skein-like inclusions, one of the two types of ubiquitinated intraneuronal inclusions in amyotrophic lateral sclerosis (ALS), in the neostriatum are not specific to the disease, but it has not yet been determined whether the other, spherical or crescent-shaped inclusions (SCI) are pathognomonic. To clarify this and also to investigate whether the distribution of SCI in particular brain regions is associated with clinical parameters, we examined the occurrence of SCI in the brains of 24 patients with ALS and 94 controls. SCI in the neostriatum were specifically detected in 54% of the ALS cases, but not in any of the controls. No apparent phenotypic denominator, such as disease duration or the occurrence of dementia, correlated to the distribution of SCI in the neostriatum in ALS cases. On the other hand, the occurrence of SCI in both the second and third layers of the parahippocampal gyrus and amygdala was significantly correlated to the presence of dementia in ALS cases. SCI were distributed in association with each other among the parahippocampal gyrus, dentate gyrus of the hippocampus and amygdala, but not between the spinal anterior horn and any non-motor-associated brain regions. These findings suggest that these particular brain regions might be significantly involved in the neurodegenerative process associated with ALS. The relationship of SCI to either ALS pathogenesis or cognitive dysfunction depends on the brain regions in which they are distributed, and this indicates that the neurodegenerative processes in ALS proceed differentially in particular motor-associated and nonmotor-associated brain regions.

10] In situ methods for detection and localization of markers of oxidative stress: Application in neurodegenerative disorders

Publisher Summary Despite an extensive research effort aimed at characterizing the nature of protein abnormalities associated with age-related neurodegenerative conditions, the factors leading to the deposition of these protein aggregates remain elusive. Neurofibrillary tangles (NFT) and senile plaques (SP) in Alzheimer's disease (AD), intraneuronal filamentous inclusions in amyotrophic lateral sclerosis (ALS), Lewy bodies in Parkinson's disease (PD), and Pick bodies in Pick's disease are pathological hallmarks. These deposits consist of one or more proteins, including amyloid- β (A β ), neurofilaments (NF), and microtubule-associated protein τ, which, in one or another form, are present in nonaggregated forms in normal cells. The various deposits also differ in terms of the degree to which aggregation is reversible and the conditions needed to achieve their solubilization, thereby complicating efforts aimed at direct structural analysis. Various factors have been proposed as the initiating events in pathological aggregation and include abnormal levels of protein synthesis, abnormal precursor protein processing, and abnormal phosphorylation or other post translational modification. In no case, however, is there a clear mechanistic consensus nor is there any direct evidence that connects protein deposition with the etiology of neuronal death in the respective neurodegenerative disease. Advances in molecular genetics and neurochemistry have associated neuronal death at least indirectly to oxidative stress, the condition when pro-oxidant events outweigh the cellular antioxidant defenses. In oxidative stress, a compromise in the normal physiological removal of reactive oxygen species (ROS) and/or an increase in their production results in irreversible damage to biomacromolecules.

Ubiquitinated Dot-Profiles (UDP) are decreased in the hippocampus in Alzheimer's disease compared with Lewy body dementia, Parkinson's disease, and controls : R Raghavan, PG Ince, RH Perry. Neuropathology Department, Newcastle General Hospital, Newcastle upon Tyne NE4 6BE UK

Ubiquitinated cytoplasmic inclusions are a characteristic feature of the neuronal pathology of neurodegenerative diseases. Immunocytochemical techniques have identified intermediate filaments associated with ubiquitin-immunoreactive inclusions in Alzheimer's disease (AD), Parkinson's disease (PD), and Pick's disease; however, no core protein has been detected in the ubiquitinated inclusions in amyotrophic lateral sclerosis (ALS). The pathogenesis of these inclusions is not known, but the inclusion may result from an accumulation of an abnormal proteins. Here we report a novel protein of 32.5 kDa detected by polyacrylamide gel electrophoresis, in the spinal cord in ALS patients. A polyclonal antibody raised against this protein and used for Western blotting, suggests that the novel protein is related to actin. Immunocytochemical studies using this antibody indicate that the protein is found in Lewy body-like inclusions in anterior horn cells of ALS, and in Lewy bodies in the substantia nigra in PD.

Antibody to an abnormal protein in amyotrophic lateral sclerosis identifies Lewy body-like inclusions in ALS and Lewy bodies in Parkinson's disease

Ubiquitinated cytoplasmic inclusions are a characteristic feature of the neuronal pathology of neurodegenerative diseases. Immunocytochemical techniques have identified intermediate filaments associated with ubiquitin-immunoreactive inclusions in Alzheimer's disease (AD), Parkinson's disease (PD), and Pick's disease; however, no core protein has been detected in the ubiquitinated inclusions in amyotrophic lateral sclerosis (ALS). The pathogenesis of these inclusions is not known, but the inclusion may result from an accumulation of an abnormal proteins. Here we report a novel protein of 32.5 kDa detected by polyacrylamide gel electrophoresis, in the spinal cord in ALS patients. A polyclonal antibody raised against this protein and used for Western blotting, suggests that the novel protein is related to actin. Immunocytochemical studies using this antibody indicate that the protein is found in Lewy body-like inclusions in anterior horn cells of ALS, and in Lewy bodies in the substantia nigra in PD.

Hyaline and Skein‐like Inclusions in Amyotrophic Lateral Sclerosis

Hyaline (HI) and skein‐like inclusions (SLI) are characteristic of familial and sporadic amyotrophic lateral sclerosis (ALS). The HIs are usually round eosinophilic structures, some of which consist of cores and halos and are called Lewy body‐like HIs. The HIs, composed of various amounts of granule‐associated thick filaments and neurofilaments, have varied appearances and may be heterogeneous. The SLI is basically a thready structure of a bundle of thick filaments. The threads may be present singly or in the form of networks or aggregates with each other. Occasionally, aggregates of threads may form single larger inclusions that resemble HIs. These features indicate that the HI and the SLI may be closely related. Other than their intense ubiquitination, the molecular nature of HI and SLI is still unknown. The investigation of the genesis of these inclusions may provide important clues for elucidating the pathomechanism of ALS.

Histochemical and immunocytochemical study of ubiquitinated neuronal inclusions in amyotrophic lateral sclerosis

Ubiquinated cytoplasmic inclusions are a characteristic feature of the anterior horn cell pathology of amyotrophic lateral sclerosis. The underlying abnormality leading to the production of these inclusions in this neurodegenerative motor system disease is unknown. Despite the application of a wide range of histochemical and immunocytochemical techniques we have been unable to identify a core constituent protein in these intraneuronal inclusions. A novel approach to this problem is required.

Hippocampal and neocortical ubiquitin-immunoreactive inclusions in amyotrophic lateral sclerosis with dementia

Amyotrophic lateral sclerosis (ALS) patients with dementia were found to have ubiquitin-immunoreactive (IR) inclusions in the dentate granule cells of the hippocampus. These inclusions were also present in some patients with minor cognitive changes but otherwise typical ALS. Ubiquitin-IR inclusions were also found in neurons of superficial layers of the frontal and temporal cortex and in the entorhinal cortex in patients with ALS and dementia. These ubiquitin-IR inclusions were non-argyrophilic, and were not labelled by antibodies which identify Alzheimer's neurofibrillary tangles and Pick bodies, nor were they typical of cortical Lewy bodies. Our findings indicate that ubiquitin-IR inclusions in small neurons of the hippocampus, entorhinal area and neocortex are a characteristic feature of degeneration of non-motor cortex in ALS, and are particularly associated with cognitive impairment and dementia of frontal lobe type.

Chronic Aluminum-Induced Motor Neuron Degeneration: Clinical, Neuropathological and Molecular Biological Aspects

The monthly intracisternal inoculation of young adult New Zealand white rabbits with low-dose (100 micrograms) aluminum chloride induces aggregates of phosphorylated neurofilament that mimics the intraneuronal inclusions of amyotrophic lateral sclerosis. The chronic progressive myelopathy and topographically-specific motor neuron degeneration that occurs in the absence of suppressions of neurofilament messenger RNA levels in this model contrasts with the acute fulminant encephalomyelopathy and nonspecific gene suppressions that occur subsequent to high-dose (1000 micrograms) aluminum chloride inoculations. Further analysis of this unique model of chronic motor system degeneration can be expected to provide additional insights into the pathogenesis of amyotrophic lateral sclerosis.

Neurofibrillary changes in human brain. An immunocytochemical study with a neurofilament antiserum.

Brain samples from cases of Alzheimer's disease, postencephalitic Parkinson's disease, progressive supranuclear palsy, amyotrophic lateral sclerosis, and Pick's disease, as well as from a case of Alzheimer's disease with a large number of Hirano bodies, were stained with the peroxidase-anti-peroxidase method using an antiserum previously shown to immunoreact with normal neurofilaments and neurofilament polypeptides. The specificity of this serum was confirmed by absorption an purified neurofilament proteins. Neurofibrillary tangles of Alzheimer's disease, postencephalitic Parkinson's disease, and progressive supranuclear palsy, Pick's bodies, and the fibrillary inclusions of amyotrophic lateral sclerosis were all immunostained. Hirano bodies showed no immunostaining. Thus, with the exception of the Hirano bodies, all the neuronal fibrillary inclusions examined appeared to share common antigenic characteristics. The orgin of all these structures from normal neurofilaments is postulated.