Skein-like Inclusions Research Articles

Neuropathology of the Neurodegenerative Diseases

A definite diagnosis of neurodegenerative diseases is required for neuropathological examination during an autopsy. Each neurodegenerative disease has specific vulnerable regions and affected systems (system degeneration), and is typified by an accumulation of abnormal protein with the formation of characteristic morphological aggregates in the nerve and glial cells, called proteinopathy. The most common neurodegenerative diseases are tauopathy, such as progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), and Pick's disease (PiD); α-synucleinopathy, including multiple system atrophy (MSA); and TAR DNA-binding protein of 43 kDa (TDP-43) proteinopathy, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). PSP and CBD show characteristic tau-positive astrocytic inclusions known as tufted astrocytes and astrocytic plaques, respectively. PiD shows tau-positive neuronal inclusions termed Pick bodies. MSA is characterized by α-synuclein-positive oligodendroglial inclusions, called glial cytoplasmic inclusions. ALS- and FTLD-TDP show TDP-43-positive neuronal inclusions, such as skein-like and round inclusions. Huntington's disease shows polyglutamine-positive neuronal inclusions, and Creutzfeldt-Jakob disease shows diffuse deposition of granular prions in the neuropil. The atypical proteins in these diseases have abnormal conformational properties. A comprehensive comparison of the clinical findings and neuropathological observations, including neuroanatomy and images acquired during life, is important to improve the sensitivity of clinical diagnosis.

Nuclear Expression of TDP-43 Is Linked with Morphology and Ubiquitylation of Cytoplasmic Aggregates in Amyotrophic Lateral Sclerosis.

The transactive response DNA-binding protein of 43 kDa (TDP-43) is a pathological protein of amyotrophic lateral sclerosis (ALS). TDP-43 pathology is characterized by a combination of the cytoplasmic aggregation and nuclear clearance of this protein. However, the mechanisms underlying TDP-43 pathology have not been fully clarified. The aim of this study was to evaluate the relationships between the expression level of nuclear TDP-43 and the pathological properties of cytoplasmic aggregates in autopsied ALS cases. We included 22 consecutively autopsied cases with sporadic TDP-43-related ALS. The motor neuron systems were neuropathologically assessed. We identified 790 neurons with cytoplasmic TDP-43 inclusions from the lower motor neuron system of included cases. Nuclear TDP-43 disappeared in 84% (n = 660) and expressed in 16% (n = 130) of neurons with cytoplasmic inclusions; the former was defined as TDP-43 cytoplasmic immunoreactivity (c-ir), and the latter was defined as nuclear and cytoplasmic immunoreactivity (n/c-ir). Morphologically, diffuse cytoplasmic inclusions were significantly more prevalent in TDP-43 n/c-ir neurons than in c-ir neurons, while skein-like and round inclusions were less prevalent in n/c-ir neurons. The cytoplasmic inclusions of TDP-43 n/c-ir neurons were phosphorylated but poorly ubiquitylated when compared with those of c-ir neurons. TDP-43 n/c-ir neurons became less dominant than the c-ir neurons among cases with a prolonged disease duration. The expression level of nuclear TDP-43 was significantly lower in n/c-ir neurons than in normal neurons without cytoplasmic inclusions. Our results indicate that the maturation of cytoplasmic TDP-43 inclusions correlates with the depletion of nuclear TDP-43 in each affected neuron. This finding supports the view that an imbalance between nuclear and cytoplasmic TDP-43 may be an essential pathway to TDP-43 pathology.

Accumulation of Nonfibrillar TDP-43 in the Rough Endoplasmic Reticulum Is the Early-Stage Pathology in Amyotrophic Lateral Sclerosis.

Transactivation response DNA-binding protein 43 (TDP-43)-immunoreactive neuronal cytoplasmic inclusions (NCIs) are the histopathological hallmarks of amyotrophic lateral sclerosis (ALS). They are classified as skein-like inclusions, round inclusions, dot-like inclusions, linear wisps, and diffuse punctate cytoplasmic staining (DPCS). We hypothesized that TDP-43-immunoreactive DPCS may form the early-stage pathology of ALS. Hence, we investigated phosphorylated TDP-43 pathology in the upper and lower motor neurons of patients with ALS and control participants. We designated patients whose disease duration was ≤1 year as short-duration ALS (n = 7) and those whose duration equaled 3-5 years as standard-duration ALS (n = 6). DPCS and skein-like inclusions were the most common NCIs in short-duration and standard-duration ALS, respectively. The density of DPCS was significantly higher in short-duration ALS than that in standard-duration ALS and was inversely correlated with disease duration. DPCS was not ubiquitinated and disappeared after proteinase K treatment, suggesting that it was not aggregated. Immunoelectron microscopy revealed that DPCS corresponded to nonfibrillar TDP-43 localized to the ribosomes of the rough endoplasmic reticulum (ER). These findings suggest that nonfibrillar TDP-43 accumulation in the rough ER is the earliest TDP-43 pathology in ALS, which may be helpful in developing future TDP-43 breakdown strategies for ALS.

Transactivation response DNA-binding protein of 43 kDa proteinopathy and lysosomal abnormalities in spastic paraplegia type 11.

Spastic paraplegia type 11 (SPG11) is the most common autosomal recessive hereditary spastic paraplegia with thinning of the corpus callosum. Spatacsin, a protein encoded by the SPG11 gene, is associated with autophagy. SPG11 patients show spastic paraplegia, intellectual disability, dementia, and parkinsonism. A previous neuropathological analysis of SPG11 cases reported neurodegeneration mimicking amyotrophic lateral sclerosis without transactivation response DNA-binding protein of 43 kDa (TDP-43) deposits and unique sequestosome 1 (SQSTM1)-positive neuronal inclusions. We performed a neuropathological examination of two Japanese patients with complicated spastic paraplegia with thinning of the corpus callosum from different families, and one was genetically diagnosed as having SPG11. Both cases showed diffuse atrophy of the brain and spinal cord. Depigmentation of the substantia nigra was also observed. Immunohistochemistry revealed widespread distribution of areas showing TDP-43 aggregation in the central nervous system. The TDP-43 deposits in the thalamus and substantia nigra especially resembled skein-like inclusions. Unique SQSTM1-positive neuronal inclusions, as previously reported, were widespread in the whole central nervous system as well as the dorsal root ganglia. Double-labeling immunofluorescence of the dorsal root ganglia revealed that the unique, large SQSTM1-positive cytoplasmic inclusions of the ganglion cells were labeled with lysosome-associated membrane protein 1 and lysosome-associated membrane protein 2. This is the first report showing TDP-43 pathology in SPG11. The common neuropathological findings of TDP-43-positive inclusions in both the cases imply a causal connection between the TDP-43 proteinopathy and autophagy dysfunction in SPG11.

Human Ubiquilin 2 and TDP-43 copathology drives neurodegeneration in transgenic Caenorhabditis elegans.

Amyotrophic lateral sclerosis (ALS) is a debilitating, fatal neurodegenerative disease that causes rapid muscle wasting. It shares a spectrum of symptoms and pathology with frontotemporal lobar degeneration (FTLD). These diseases are caused by aberrant activity of a set of proteins including TDP-43 and UBIQUILIN-2 (UBQLN2). UBQLN2 encodes a ubiquitin-like adaptor protein involved in the ubiquitin-proteasome protein degradation pathway. Mutations in the PXX domain of UBQLN2 cause familial ALS. UBQLN2 aggregates in skein-like inclusions with other ALS and FTLD associated proteins including TDP-43 and ubiquitin. To facilitate further investigation of UBQLN2-mediated mechanisms of neurodegeneration, we made Caenorhabditis elegans transgenic lines pan-neuronally expressing human UBQLN2 cDNAs carrying either the wild-type UBQLN2 sequence or UBQLN2 with ALS causing mutations. Transgenic animals exhibit motor dysfunction accompanied by neurodegeneration of GABAergic motor neurons. At low levels of UBQLN2 expression, wild-type UBQLN2 causes significant motor impairment and neurodegeneration that is exacerbated by ALS associated mutations in UBQLN2. At higher levels of UBQLN2 expression, both wild-type and ALS mutated versions of UBQLN2 cause severe impairment. Molecular genetic investigation revealed that UBQLN2 dependent locomotor defects do not require the involvement of the endogenous homolog of TDP-43 in C. elegans (tdp-1). However, co-expression of wild-type human TDP-43 exacerbates UBQLN2 deficits. This model of UBQLN2-mediated neurodegeneration may be useful for further mechanistic investigation into the molecular cascades driving neurodegeneration in ALS and ALS-FTLD.

Aggresome formation and liquid\u2013liquid phase separation independently induce cytoplasmic aggregation of TAR DNA-binding protein 43

Cytoplasmic inclusion of TAR DNA-binding protein 43 (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and a subtype of frontotemporal lobar degeneration (FTLD). Recent studies have suggested that the formation of cytoplasmic TDP-43 aggregates is dependent on a liquid–liquid phase separation (LLPS) mechanism. However, it is unclear whether TDP-43 pathology is induced through a single intracellular mechanism such as LLPS. To identify intracellular mechanisms responsible for TDP-43 aggregation, we established a TDP-43 aggregation screening system using a cultured neuronal cell line stably expressing EGFP-fused TDP-43 and a mammalian expression library of the inherited ALS/FTLD causative genes, and performed a screening. We found that microtubule-related proteins (MRPs) and RNA-binding proteins (RBPs) co-aggregated with TDP-43. MRPs and RBPs sequestered TDP-43 into the cytoplasmic aggregates through distinct mechanisms, such as microtubules and LLPS, respectively. The MRPs-induced TDP-43 aggregates were co-localized with aggresomal markers and dependent on histone deacetylase 6 (HDAC6), suggesting that aggresome formation induced the co-aggregation. However, the MRPs-induced aggregates were not affected by 1,6-hexanediol, an LLPS inhibitor. On the other hand, the RBPs-induced TDP-43 aggregates were sensitive to 1,6-hexanediol, but not dependent on microtubules or HDAC6. In sporadic ALS patients, approximately half of skein-like TDP-43 inclusions were co-localized with HDAC6, but round and granular type inclusion were not. Moreover, HDAC6-positive and HDAC6-negative inclusions were found in the same ALS patient, suggesting that the two distinct pathways are both involved in TDP-43 pathology. Our findings suggest that at least two distinct pathways (i.e., aggresome formation and LLPS) are involved in inducing the TDP-43 pathologies.

Autophagy Is a Common Degradation Pathway for Bunina Bodies and TDP-43 Inclusions in Amyotrophic Lateral Sclerosis

Bunina bodies (BBs) coexisting with TDP-43-immunoreactive (TDP-43-IR) skein-like inclusions (SIs) and round inclusions (RIs) in lower motor neurons are a frequent feature of sporadic amyotrophic lateral sclerosis (sALS). Since previous studies have shown that BBs and TDP-43-IR inclusions are often detected in association with autophagy-related structures (autophagosomes and autolysosomes), we examined the anterior horn cells (AHCs) of the spinal cord from 15 patients with sALS and 6 control subjects, using antibodies against autophagy-related proteins (LC3, cathepsin B, and cathepsin D). Among AHCs with SIs, 43.9% contained BBs, whereas 51.7% of AHCs with RIs did so. The cytoplasm of AHCs showed diffuse immunoreactivity for LC3, cathepsin B and cathepsin D in both sALS and controls. Ultrastructurally, SIs and mature BBs contained autophagosomes and autolysosomes. Mature BBs were localized in the vicinity of SIs. RIs also contained autophagosomes, autolysosomes, and early-stage BBs. These findings suggest that autophagy is a common degradation pathway for BBs and TDP-43-IR inclusions, which may explain their frequent coexistence.

Anterior Cingulate Cortex TDP-43 Pathology in Sporadic Amyotrophic Lateral Sclerosis.

Neuronal constituents of the human anterior cingulate cortex displayed morphological changes related to the 43-kDa transactive response DNA-binding protein (TDP-43) in advanced pathological stages of sporadic amyotrophic lateral sclerosis (sALS). By using nonphosphorylation-dependent TDP-43 immunocytochemistry, it was seen that the changes in susceptible pyramidal cells of the superficial cellular layers II-IIIab differed from those in the deep layers IIIc-Vb: A complete loss of nuclear TDP-43 expression (i.e. nuclear clearing) in the small projection neurons of layers II-IIIab was consistently accompanied by the development of somatic skein-like TDP-43-immunopositive inclusions. In contrast, in the large pyramidal cells of layers IIIc-Vb and von Economo neurons of layer Vb, skein-like inclusions were lacking or, when aggregated TDP-43 was present, the aggregates presented as dash-like TDP-43-immunopositive particles in the vicinity of the cell nucleus. The cytoskeleton of projection neurons in layers II-IIIab is neurofilament-sparse in contrast to that of the large neurons in layers IIIc-Vb, which are rich in neurofilaments and also heat shock proteins that function as their molecular chaperones. The disparities between the two neuronal populations may contribute to the two differing immunocytochemical profiles reported here. Some implications of the findings for the pathogenesis and progression of TDP-43 pathology in sALS are discussed.

Hypersialylation is a common feature of neurofibrillary tangles and granulovacuolar degenerations in Alzheimer's disease and tauopathy brains.

Glycosylation is one of the major post-translational modifications of proteins. The status of sialylation of the neuropathological hallmarks of various neurodegenerative disorders was investigated in this study. Here, we report the novel findings that two phosphorylated tau (p-tau)-containing structures associated with Alzheimer's disease (AD), that is, neurofibrillary tangles (NFTs) and granulovacuolar degenerations (GVDs), were hypersialylated. The NFTs, GVDs and dystrophic neurites of senile plaques (SPs) in AD hippocampi were clearly visualized by immunohistochemistry using an anti-sialic acid (SA) antibody. In contrast, the amyloid core of SPs was not sialylated at all. Interestingly, other p-tau-containing structures, that is, globose-type NFTs in progressive supranuclear palsy and Pick bodies and ballooned neurons in frontotemporal lobar degeneration with Pick bodies, were also hypersialylated. Unlike the p-tau-containing structures observed in tauopathies, the hallmarks of other neurodegenerative disorders, such as Lewy bodies in Parkinson's disease, glial cytoplasmic inclusions in multiple system atrophy, Bunina bodies, skein-like inclusions and round inclusions in amyotrophic lateral sclerosis, intranuclear inclusions in neuronal intranuclear inclusion disease and physiological bodies or granules (lipofuscin granules, corpora amylacea and melanin granules), were not immunolabeled by the anti-SA antibody. Because this antibody specifically identified NFTs and GVDs, immunostaining for sialylation represents a useful tool to screen these structures in a diagnostic setting. These results clearly indicate that the pathological hallmarks of various tauopathies are commonly hypersialylated, and that sialylation plays an important role in the process of p-tau accumulation in AD and other tauopathies.

Loss of hnRNPA1 in ALS spinal cord motor neurons with TDP-43-positive inclusions.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by loss of motor neurons and appearance of skein-like inclusions. The inclusions are composed of trans-activation response (TAR) DNA-binding protein 43 (TDP-43), a member of the heterogeneous nuclear ribonucleoprotein (hnRNP) family. hnRNPA1 and hnRNPA2/B1 are hnRNPs that interact with the C-terminus of TDP-43. Using immunohistochemistry, we investigated the association between TDP-43 and hnRNPA1 in ALS spinal motor neurons. We examined spinal cords of seven ALS cases and six muscular dystrophy cases (used as controls) for the presence of TDP-43 and hnRNPA1 protein. In the control cases, hnRNPA1 immunoreactivity in motor neurons was intense in the nucleus and weak in the cytoplasm where it showed a fine granular appearance. In the ALS cases, hnRNPA1 immunoreactivity in motor neurons was reduced in the nuclei of neurons with skein-like inclusions but was not detected in the skein-like inclusions. The marked loss of hnRNPA1 in motor neurons with concomitant cytoplasmic aggregation of TDP-43 may represent a severe disturbance of mRNA processing, suggesting a key role in progressive neuronal death in ALS.

AMYOTROPHIC LATERAL SCLEROSIS ASSOCIATED WITH AN INTERMEDIATE LENGTH GGGGCC REPEAT EXPANSION HAS DISTINCT NEUROPATHOLOGY COMPARED TO PATIENTS WITH LARGER EXPANSIONS

<h3>Background</h3> An intronic GGGGCC hexanucleotide repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Thirty repeats is the suggested pathogenic cut-off. However, the 9p21 risk haplotype has been associated with C9orf72 expansions &gt;7 repeats, leading to the description of 7–24 repeats as intermediate. The pathogenicity of intermediate repeat lengths is unknown. Studies have suggested phenotypic similarities between patients with intermediate length expansions and those with larger repeat lengths, although given the broad phenotypic spectrum of ALS this is not conclusive. Previously, there has been no pathological characterisation of an intermediate expansion patient. <h3>Materials and Methods</h3> Pathological material was obtained from the Sheffield Brain Tissue Bank. Genomic DNA was extracted from cerebellar material and reverse primed PCR and Southern hybridisation was conducted to size the expansion. Immunohistochemistry and RNA fluorescence in-situ hybridisation (FISH) was used to screen for pathology typical of C9orf72-ALS. <h3>Results</h3> Repeat-primed PCR and Southern hybridisation revealed a repeat expansion size of 16 repeats in a sporadic ALS (SALS) patient. Neuropathological analysis demonstrated loss of motor neurons and immunohistochemistry revealed coarse neuronal skein-like inclusions identified by ubiquitin and P62 immunostaining. Pathology characteristic of C9orf72-disease, including extramotor neuronal inclusions, RNA foci and neuronal inclusions containing poly-(Gly-Ala)- dipeptide repeat protein, were absent in this patient. <h3>Conclusion</h3> A patient with an intermediate C9orf72 expansion of 16 repeats did not show the characteristic C9orf72 pathology suggesting that, in this case, the disease process was distinct from C9orf72-ALS, and the intermediate expansion is an incidental finding. This is consistent with current toxic gain-of-function theories of C9orf72-ALS. The 9p21 risk haplotype may predispose to expansion of the GGGGCC repeat locus, but we suggest that this must occur to some length larger than 16 repeats to cause the pathology characteristic of the C9orf72 subtype of ALS.

Cytoplasmic TDP-43 accumulation in cells of the adrenal medulla in individuals with or without amyotrophic lateral sclerosis.

The transactive response DNA-binding protein of 43 kDa (TDP-43) is normally located predominantly in the nucleus, whereas pathological TDP-43 is mostly found in the cytoplasm. Cytoplasmic TDP-43 accumulation has not yet been reported in normal general organs. In our preliminary study, paraffin sections of the general organs of individuals with or without amyotrophic lateral sclerosis (ALS) were immunostained with antibodies against TDP-43 and phosphorylated TDP-43 (pTDP-43). Diffuse and granular immunostaining pattern of TDP-43 and pTDP-43 were observed frequently in the cytoplasm of renal tubular cells, and less frequently in the cells of several organs; however, the majority of these immunoreactivities were nonspecific biotin reactions. Conversely, many TDP-43-positive and pTDP-43-negative cytoplasmic accumulations were observed in the adrenal medulla in every individual (with or without ALS). Skein-like or round inclusions were not observed. The cells in the adrenal medulla were well preserved, and the cytoplasmic TDP-43 accumulations were frequent in the cells of all routine autopsy cases without loss of nuclear TDP-43 immunostaining; therefore, we considered that this was a physiological phenomenon. This is the first study that demonstrated the cytoplasmic accumulation of TDP-43 in routinely autopsied cases.

Lower Motor Neuron Involvement in TAR DNA-Binding Protein of 43 kDa–Related Frontotemporal Lobar Degeneration and Amyotrophic Lateral Sclerosis

TAR DNA-binding protein of 43 kDa (TDP-43) plays a major role in the pathogenesis of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Although a pathological continuity between FTLD and ALS has been suggested, the neuropathological changes of the lower motor neuron (LMN) systems have not been assessed in TDP-43-associated FTLD (FTLD-TDP), to our knowledge. To investigate a pathological continuity between FTLD-TDP and ALS by comparing their respective neuropathological changes in the motor neuron system. A retrospective clinical medical record review and a semiquantitative neuropathological evaluation of the cranial motor nerve nuclei and spinal cord were conducted at autopsy. We included 43 patients with sporadic FTLD-TDP, type A, B, or C, from 269 consecutively autopsied patients with TDP-43 proteinopathy. Patients were categorized as having FTLD without ALS, FTLD-ALS (onset of FTLD symptoms/signs preceded those of ALS), or ALS-FTLD (onset of ALS symptoms/signs preceded those of FTLD). Neuronal TDP-43 pathological changes and neuronal loss. Forty-three patients were included in the clinical analysis, and 29 from whom spinal cords were obtained were included in the neuropathological analysis. Survival time was significantly shorter in the FTLD-ALS and ALS-FTLD groups than in the FTLD without ALS group (P < .001). At neuropathological examination, 89% of patients in the FTLD without ALS group showed aggregations of TDP-43 in the spinal motor neurons. The LMN loss was most severe in ALS-FTLD, followed by FTLD-ALS and FTLD without ALS. All the patients with type A or C FTLD-TDP were included in the FTLD without ALS group, and all those with type B pathological changes were in the FTLD-ALS or the ALS-FTLD group. Lower motor neuron loss and TDP-43-positive skeinlike inclusions were observed in all pathological subtypes. The LMN systems of FTLD-TDP frequently exhibit neuropathological changes corresponding to ALS. Thus, a pathological continuity between FTLD-TDP and ALS is supported at the level of the LMN system.

Coexistence of amyotrophic lateral sclerosis with neuro‐Behçet's disease presenting as a longitudinally extensive spinal cord lesion: clinicopathologic features of an autopsied patient

We report the clinical and autopsy features of a 65-year-old Japanese man who clinically exhibited overlap of both neuro-Behçet's disease (NBD) and amyotrophic lateral sclerosis (ALS). The patient had a HLA-B51 serotype, a recent history of uveitis and had suffered paraparesis, sensory and autonomic disturbance, frontal signs and tremor. A brain and spine MRI study revealed a longitudinally extensive thoracic cord (Th) lesion, but no apparent intracranial abnormalities. The lesion extended ventrally from Th4 to Th9, exhibiting low intensity on T1-weighted images, high intensity on T2-weighted and fluid-attenuated inversion recovery images and gadolinium enhancement. The patient's upper and lower motor neuron signs and sensory disturbance worsened and he died 16 months after admission. At autopsy, the spinal cord and brain exhibited characteristic histopathological features of both NBD and ALS, including chronic destruction of the ventral thoracic white and gray matter, perivascular lymphocytic infiltration, binucleated neurons, lower and upper motor neuron degeneration, Bunina bodies and skein-like inclusions. Although incidental coexistence of these rare disorders could occur in an individual, this case raises the possibility of a pathomechanistic association between NBD and ALS.

TAR DNA-binding protein 43 pathology in a case clinically diagnosed with facial-onset sensory and motor neuronopathy syndrome: An autopsied case report and a review of the literature

We report an autopsy case of a 48-year-old female clinically diagnosed with facial-onset sensory and motor neuronopathy (FOSMN) syndrome with TAR DNA-binding protein 43 (TDP-43) pathology. She developed paresthesia involving her whole face, right upper extremity and the right side of her upper trunk, followed by dysphagia, dysarthria, muscle atrophy and weakness with fasciculation in both upper extremities. Her symptoms showed a marked cranial and right-sided dominancy. She had anti-sulfoglucuronyl paragloboside (SGPG) IgG and anti-myelin-associated glycoprotein (MAG) IgG, and repeatedly showed limited response to immunotherapies. Her disease was essentially progressive, culminating in death due to respiratory failure three and a half years after onset. The autopsy revealed severe degeneration of the nuclei of the right trigeminal nerve and right facial nerve and widespread TDP-43-positive glial inclusions in the brainstem tegmentum. Neurons in the hypoglossal nerve nuclei were also shrunken and lost, with TDP-43-positive neuronal inclusions. Neuronal loss and gliosis in the anterior horn, predominantly in the cervical cord, were prominent with TDP-43-positive skein-like inclusions. Bilateral ventral roots were obviously atrophic. Spinal tract degeneration was also prominent in the ventral columns, essentially sparing the anterior corticospinal tracts at the cervical cord level. Additionally there was severe myelin pallor in the right spinal trigeminal tract and right fasciculus cuneatus of the cervical cord. The right spinal root ganglion showed numerous Nageotte's nodules and focal lymphocytic infiltration. The present case manifested FOSMN syndrome clinically, while the pathological findings suggested a motor neuron disease like TDP-43 proteinopathy and a possible involvement of immune-mediated neuropathy.

An autopsy case of sporadic amyotrophic lateral sclerosis associated with the I113T SOD1 mutation

A 64-year-old man noticed weakness in his arms and dyspnea upon exertion. Four months later he was admitted to our hospital, where muscle atrophy and hyperactive deep tendon reflexes in the arms were observed upon examination. A needle electromyograph study revealed acute and chronic denervation in the extremities, and he was diagnosed as having amyotrophic lateral sclerosis (ALS). Seven months after onset of the disease, he died of respiratory failure. Neuropathologically, neuronal cell loss was observed in the motor cortex, hypoglossal nuclei, cervical and lumbar anterior horns and Clarke's nuclei. Some of the remaining neurons contained neurofilamentous conglomerate inclusions (CIs). A small number of Lewy body-like hyaline inclusions (LBHIs) were also observed. No the Bunina bodies, skein-like inclusions or basophilic inclusions were detectable. Tract degeneration was moderate in the dorsal and ventral spinocerebellar tracts, mild in the pyramidal tract, but not discerned in the posterior column. Immunohistochemical examinations revealed that the CIs were strongly positive for phosphorylated neurofilament and moderately positive for ubiquitin and Cu/Zn superoxide dismutase 1 (SOD1). Moreover, a number of phosphorylated tau protein-positive globose neurofibrillary tangles (NFTs) and threads were observed in the periaqueductal gray matter, oculomotor nuclei and trochlear nuclei. Although the family history was negative for neuromuscular diseases, the neuropathological findings indicated features of familial ALS with a SOD1 mutation. In fact, DNA analysis of frozen-brain tissue revealed the presence of the I113T SOD1 mutation. This case represents the first one of this mutation in a patient who showed CIs as well as LBHIs in the motor neurons at the same time, in addition to the NFTs in the mesencephalic tegmentum.

Inclusions in frontotemporal lobar degeneration with TDP-43 proteinopathy (FTLD-TDP) and amyotrophic lateral sclerosis (ALS), but not FTLD with FUS proteinopathy (FTLD-FUS), have properties of amyloid

TDP-43 and FUS normal cytoplasmic functions are thought to involve regulated aggregation and disaggregation [1, 5, 8], similar to that of prion proteins, where aggregation occurs by a self-templating process, likely involving properties of amyloid, or beta pleated sheet structure. Both thioflavin-T and thioflavin-S fluoresce when bound to amyloid fibrils [7]. We previously showed that TDP-43 peptides form amyloidogenic fibrils, binding to thioflavin-T [3]. Recently, TDP-43-positive lower motor neuron (LMN) inclusions in 28% of 47 cases of ALS, but no inclusions in 22 FTLD-TDP cases, were shown to be positive with thioflavin-S [6]. Using thioflavin-S, we surveyed brain tissues from 44 cases, including FTLD-TDP type A, (17 cases), type B (14 cases), type C (3 cases), sporadic ALS (2 cases) familial ALS (FALS) (2 each with SOD1 and C9orf72 mutations), and FTLD-FUS, including atypical FTLD-U (aFTLD-U, 2 cases) and basophilic inclusion body disease (BIBD, 2 cases). Our routine modified thioflavin-S stain includes pretreatment of tissue sections with potassium permanganate and bleaching with potassium metabisulfite and oxalic acid followed by treatment with sodium hydroxide and hydrogen peroxide, removing lipid autofluorescence and resulting in improved definition of pathological lesions. Slides were viewed using a Nikon BV-2A filter cube. Confocal microscopy was performed using thioflavin-S staining as above and phospho TDP43 (pS409/410-1). Inclusions in most cases of FTLD-TDP and ALS were thioflavin-S positive. The density and distribution of thioflavin-S positive inclusions was similar to that seen with ubiquitin and fewer than with TDP-43 immunohistochemistry. In FTLD-TDP type A, neuronal intranuclear inclusions (NIIs), neuronal cytoplasmic inclusions (NCIs) and dystrophic neurites (DNs) were strongly fluorescent (Fig. 1a). Fluorescent confocal microscopy showed co-localization of thioflavin-S with TDP-43 positive inclusions (Fig. 1b). In FTLD-TDP type B, rare NCIs in all layers of cortex were positive but dentate gyrus inclusions were easily seen (Fig. 1c). In FTLD-TDP type C, long DNs in cortex were strongly thioflavin-S positive (Fig. 1d). Inclusions in FTLD-FUS were negative with thioflavin-S; while rare hippocampal dentate gyrus neurons had fluorescent granular inclusions, large cytoplasmic inclusions and intranuclear vermiform inclusions were not seen with thioflavin-S (Fig. 2). Skein-like inclusions (SLI) and Lewy-like bodies (LLB) in LMNs of ALS cases were positive with thioflavin-S (Fig. 3). The two cases of FALS with SOD1 mutations were not thioflavin-S positive. Additionally, eight of the 17 cases of FTLD-TDP type A had exuberant thioflavin-S positive astrocytosis, making identification of TDP-43 pathology problematic in five of these (Fig. 4). Fig. 1 Superficial frontal cortex, FTLD-TDP type A (1a). Numerous thioflavin-S positive c-shaped and annular NCIs and a few DNs. Inset: thioflavin-S positive NII. Thioflavin-S, 400x. Confocal microscopy (1b). Same case as Fig. 1a, with thioflavin-S (green, left), ... Fig. 2 Motor cortex, FTLD-FUS (BIBD) (2a). Large cytoplasmic upper motor neuron inclusion on left, consistent with basophilic inclusion body (BIB), negative with thioflavin-S. BIB on right immunolabeled with FUS for comparison. Thioflavin-S stain and FUS immunohistochemistry, ... Fig. 3 Lumbar anterior horn, ALS. Lower motor neurons with LLB (left) and SLIs (middle and right). Scattered shrunken neurons nearby (middle and right images) contain compressed SLIs. Thioflavin-S, 600x. Fig. 4 Superficial frontal cortex, FTLD-TDP type A. Exuberant thioflavin-S positive astrocytosis. Thioflavin-S, 200x. There are at least three possible reasons that these results differ from those of Robinson et al. The modified thioflavin-S protocol used in the current study provides results that are superior to other thioflavin-S protocols, the BV-2A filter produces the brightest images, and the brain tissue is briefly paraformaldehyde-fixed (30 hours). Fixation does not likely impact results, but we cannot exclude that possibility. Clearly more cases need to be analyzed with thioflavin-S, and that work is in progress. Nonetheless, it is important that thus far, inclusions in most cases of FTLD-TDP, but not FTLD-FUS or SOD1 FALS, are thioflavin-S positive, suggesting that the prion-like property of TDP-43 may be involved in FTLD-TDP and sporadic ALS and non-SOD1 FALS, but that this may not be the case in FTLD-FUS. The significance of the thioflavin-S positive astrocytosis in some cases of FTLD-TDP type A is currently under investigation. In addition, the fact that TDP-43 in FTLD-TDP has properties of amyloid has implications for the interpretation of amyloid imaging studies [2], as Pittsburgh Compound-B is a modified thioflavin-T derivative [4].

Keap1 Is Localized in Neuronal and Glial Cytoplasmic Inclusions in Various Neurodegenerative Diseases

Oxidative stress has been proposed as a potential mechanism for neurodegenerative diseases, such as Alzheimer disease (AD), Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS). In response to oxidative stress, the levels of numerous cytoprotective products are increased via alteration of the Kelch-like ECH-associated protein 1 (Keap1) and NF-E2-related factor 2 (Nrf2) system. One of the Nrf2 targets, p62, has been known to be incorporated into a wide spectrum of cytoplasmic inclusions in neurodegenerative diseases and interact with Keap1. However, it remains unclear whether Keap1 is associated with the pathogenesis of neurodegenerative diseases. In this study, we investigated the relationship between p62 and Keap1 in the brains of patients with AD, PD, dementia with Lewy bodies (DLB), and ALS. Biochemical analyses showed that p62 and Keap1 interacted with each other in AD and DLB brains and were extracted into similar detergent-soluble and -insoluble fractions. Pathologic examination demonstrated that anti-Keap1 antibodies immunostained Lewy bodies in PD and DLB, neurofibrillary tangles in AD, and skeinlike inclusions in ALS. Further analysis showed that the levels of common Nrf2 target genes were increased in AD compared with those in controls. However, there were no statistical significances in the levels of Nrf2 target genes in DLB relative to controls. Our pathologic and biochemical results suggest a molecular basis for stress response to be involved in the formation of cytoplasmic inclusions observed in several neurodegenerative diseases.

TDP-43 skeins show properties of amyloid in a subset of ALS cases

Aggregation of TDP-43 proteins to form intracellular inclusions is the primary pathology in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) with TDP-43 inclusions (FTLD-TDP). Histologically, in the cerebral cortex and limbic regions of affected ALS and FTLD-TDP patients, these pathologies occur as a variety of cytoplasmic, neuritic and intranuclear TDP-43 inclusions. In the spinal cord and lower brainstem of ALS patients, the lesions form cytoplasmic dashes or complex filamentous and spherical profiles in addition to skein-like inclusions (SLI). Ultrastructurally, the morphology of TDP-43 inclusions is heterogeneous but mainly composed of loose bundles of 10- to 20-nm-diameter straight filaments associated with electron-dense granular material. All of these TDP-43 inclusions are generally described as disordered amorphous aggregations unlike the amyloid fibrils that characterize protein accumulations in neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. We here report that Thioflavin-S positive SLI are present in a subset of ALS cases, while TDP-43 inclusions outside the spinal cord lack the chemical properties of amyloid. Further, we examine the differential enrichment of fibrillar profiles in SLI of ALS cases by TDP-43 immuno-electron microscopy (immuno-EM). The demonstration that pathological TDP-43 can be amyloidogenic in situ suggests the following conclusions: (1) the conformational changes associated with TDP-43 aggregation are more complex than previously thought; (2) Thioflavin-S positive SLI may be composed primarily of filamentous ultrastructures.

Coexistence of Huntington’s disease and amyotrophic lateral sclerosis: a clinicopathologic study

We report a retrospective case series of four patients with genetically confirmed Huntington's disease (HD) and sporadic amyotrophic lateral sclerosis (ALS), examining the brain and spinal cord in two cases. Neuropathological assessment included a polyglutamine recruitment method to detect sites of active polyglutamine aggregation, and biochemical and immunohistochemical assessment of TDP-43 pathology. The clinical sequence of HD and ALS varied, with the onset of ALS occurring after the mid-50's in all cases. Neuropathologic features of HD and ALS coexisted in both cases examined pathologically: neuronal loss and gliosis in the neostriatum and upper and lower motor neurons, with Bunina bodies and ubiquitin-immunoreactive skein-like inclusions in remaining lower motor neurons. One case showed relatively early HD pathology while the other was advanced. Expanded polyglutamine-immunoreactive inclusions and TDP-43-immunoreactive inclusions were widespread in many regions of the CNS, including the motor cortex and spinal anterior horn. Although these two different proteinaceous inclusions coexisted in a small number of neurons, the two proteins did not co-localize within inclusions. The regional distribution of TDP-43-immunoreactive inclusions in the cerebral cortex partly overlapped with that of expanded polyglutamine-immunoreactive inclusions. In the one case examined by TDP-43 immunoblotting, similar TDP-43 isoforms were observed as in ALS. Our findings suggest the possibility that a rare subset of older HD patients is prone to develop features of ALS with an atypical TDP-43 distribution that resembles that of aggregated mutant huntingtin. Age-dependent neuronal dysfunction induced by mutant polyglutamine protein expression may contribute to later-life development of TDP-43 associated motor neuron disease in a small subset of patients with HD.