Neuronal Cytoplasmic Inclusions Research Articles

Psychiatric symptoms and TDP-43 pathology in amyotrophic lateral sclerosis

BackgroundALS is not a pure motor neuron disease but co-occurs with cognitive impairment and psychiatric symptoms. The neuropathological origin of the psychiatric symptoms is unclear. This study examined the association between the psychiatric symptoms and neuropathology of ALS. MethodsWe investigated the clinicopathological characteristics of 15 autopsy cases of ALS, including neuronal loss, gliosis, and the burden of TDP-43 pathology. We divided TDP-43–positive structures by morphology into four categories (neuronal cytoplasmic inclusion, dystrophic neurite, dot, and glial cytoplasmic inclusion) and gave each a semiquantitative score in nine brain regions. Braak neurofibrillary tangle stage, Thal amyloid phase, Lewy-related pathology, and argyrophilic grains were also assessed. ResultsOf the 15 ALS patients, seven had presented with psychiatric symptoms and eight had not. Significantly higher TDP-43 pathology scores were found in the group with psychiatric symptoms in the temporal tip, transentorhinal cortex, entorhinal cortex, subiculum, and the hippocampal CA1 region and dentate gyrus. Cognitive impairment was not significantly associated with the degree of TDP-43 pathology. There were no significant differences in the degree of neuronal loss/gliosis or in other concurrent pathologies between patients with and without psychiatric symptoms. Morphological evaluation showed that neuronal cytoplasmic inclusions, dystrophic neurites, and dots tended to be more common in the group with psychiatric symptoms. ConclusionPsychiatric symptoms in ALS may be related to TDP-43 pathology in the perforant pathway. (224 words).

Neuropathological hallmarks in the post-mortem retina of neurodegenerative diseases

The retina is increasingly recognised as a potential source of biomarkers for neurodegenerative diseases. Hallmark protein aggregates in the retinal neuronal tissue could be imaged through light non-invasively. Post-mortem studies have already shown the presence of specific hallmark proteins in Alzheimer’s disease, primary tauopathies, synucleinopathies and frontotemporal lobar degeneration. This study aims to assess proteinopathy in a post-mortem cohort with different neurodegenerative diseases and assess the presence of the primary pathology in the retina. Post-mortem eyes were collected in collaboration with the Netherlands Brain Bank from donors with Alzheimer’s disease (n = 17), primary tauopathies (n = 8), synucleinopathies (n = 27), frontotemporal lobar degeneration (n = 8), mixed pathology (n = 11), other neurodegenerative diseases (n = 6), and cognitively normal controls (n = 25). Multiple cross sections of the retina and optic nerve tissue were immunostained using antibodies against pTau Ser202/Thr205 (AT8), amyloid-beta (4G8), alpha-synuclein (LB509), pTDP-43 Ser409/410 and p62-lck ligand (p62) and were assessed for the presence of aggregates and inclusions. pTau pathology was observed as a diffuse signal in Alzheimer’s disease, primary tauopathies and controls with Alzheimer’s disease neuropathological changes. Amyloid-beta was observed in the vessel wall and as cytoplasmic granular deposits in all groups. Alpha-synuclein pathology was observed as Lewy neurites in the retina in synucleinopathies associated with Lewy pathology and as oligodendroglial cytoplasmic inclusions in the optic nerve in multiple system atrophy. Anti-pTDP-43 generally showed typical neuronal cytoplasmic inclusion bodies in cases with frontotemporal lobar degeneration with TDP-43 and also in cases with later stages of limbic-associated TDP-43 encephalopathy. P62 showed inclusion bodies similar to those seen with anti-pTDP-43. Furthermore, pTau and alpha-synuclein pathology were significantly associated with increasing Braak stages for neurofibrillary tangles and Lewy bodies, respectively. Mixed pathology cases in this cohort consisted of cases (n = 6) with high Braak LB stages (> 4) and low or moderate AD pathology, high AD pathology (n = 1, Braak NFT 6, Thal phase 5) with moderate LB pathology, or a combination of low/moderate scores for different pathology scores in the brain (n = 4). There were no cases with advanced co-pathologies. In seven cases with Braak LB ≥ 4, LB pathology was observed in the retina, while tau pathology in the retina in the mixed pathology group (n = 11) could not be observed. From this study, we conclude that the retina reflects the presence of the major hallmark proteins associated with neurodegenerative diseases. Although low or moderate levels of copathology were found in the brains of most cases, the retina primarily manifested protein aggregates associated with the main neurodegenerative disease. These findings indicate that with appropriate retinal imaging techniques, retinal biomarkers have the potential to become highly accurate indicators for diagnosing the major neurodegenerative diseases of the brain.

Phase Separation of SARS-CoV-2 Nucleocapsid Protein with TDP-43 Is Dependent on C-Terminus Domains.

The SARS-CoV-2 nucleocapsid protein (N protein) is critical in viral replication by undergoing liquid-liquid phase separation to seed the formation of a ribonucleoprotein (RNP) complex to drive viral genomic RNA (gRNA) translation and in suppressing both stress granules and processing bodies, which is postulated to increase uncoated gRNA availability. The N protein can also form biomolecular condensates with a broad range of host endogenous proteins including RNA binding proteins (RBPs). Amongst these RBPs are proteins that are associated with pathological, neuronal, and glial cytoplasmic inclusions across several adult-onset neurodegenerative disorders, including TAR DNA binding protein 43 kDa (TDP-43) which forms pathological inclusions in over 95% of amyotrophic lateral sclerosis cases. In this study, we demonstrate that the N protein can form biomolecular condensates with TDP-43 and that this is dependent on the N protein C-terminus domain (N-CTD) and the intrinsically disordered C-terminus domain of TDP-43. This process is markedly accelerated in the presence of RNA. In silico modeling suggests that the biomolecular condensate that forms in the presence of RNA is composed of an N protein quadriplex in which the intrinsically disordered TDP-43 C terminus domain is incorporated.

Distinct ultrastructural phenotypes of glial and neuronal alpha-synuclein inclusions in multiple system atrophy.

Multiple System Atrophy is characterized pathologically by the accumulation of alpha-synuclein (aSyn) into glial cytoplasmic inclusions (GCIs). The mechanism underlying the formation of GCIs is not well understood. In this study, correlative light and electron microscopy was employed to investigate aSyn pathology in the substantia nigra and putamen of post-mortem multiple system atrophy brain donors. Three distinct types of aSyn immuno-positive inclusions were identified in oligodendrocytes, neurons and dark cells presumed to be dark microglia. Oligodendrocytes contained fibrillar GCIs that were consistently enriched with lysosomes and peroxisomes, supporting the involvement of the autophagy pathway in aSyn aggregation in multiple system atrophy. Neuronal cytoplasmic inclusions exhibited ultrastructural heterogeneity resembling both fibrillar and membranous inclusions, linking multiple systems atrophy and Parkinson's disease. The novel aSyn pathology identified in the dark cells, displayed GCI-like fibrils or non-GCI-like ultrastructures suggesting various stages of aSyn accumulation in these cells. The observation of GCI-like fibrils within dark cells suggests these cells may be an important contributor to the origin or spread of pathological aSyn in multiple system atrophy. Our results suggest a complex interplay between multiple cell types that may underlie the formation of aSyn pathology in multiple system atrophy brain and highlight the need for further investigation into cell-specific disease pathologies in multiple system atrophy.

Development and Preclinical Evaluation of a Copper-64-Labeled Antibody Targeting Glycine-Alanine Dipeptides for PET Imaging of C9orf72-Associated Amyotrophic Lateral Sclerosis/Frontotemporal Dementia.

Aggregating poly(glycine-alanine) (poly-GA) is derived from the unconventional translation of the pathogenic intronic hexanucleotide repeat expansion in the C9orf72 gene, which is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Poly-GA accumulates predominantly in neuronal cytoplasmic inclusions unique to C9orf72 ALS/FTD patients. Poly-GA is, therefore, a promising target for PET/CT imaging of FTD/ALS to monitor disease progression and therapeutic interventions. A novel 64Cu-labeled anti-GA antibody (mAb1A12) targeting the poly-GA protein was developed and evaluated in a transgenic mouse model. It was obtained with high radiochemical purity (RCP), radiochemical yield (RCY), and specific activity, and showed high stability in vitro and ex vivo and specifically bound to poly-GA. The affinity of NODAGA-mAb1A12 for poly-GA was not affected by this modification. [64Cu]Cu-NODAGA-mAb1A12 was injected into transgenic mice expressing GFP-(GA)175 in excitatory neurons driven by Camk2a-Cre and in control littermates. PET/CT imaging was performed at 2, 20, and 40 h post-injection (p.i.) and revealed a higher accumulation in the cortex in transgenic mice than in wild-type mice, as reflected by higher standardized uptake value ratios (SUVR) using the cerebellum as the reference region. The organs were isolated for biodistribution and ex vivo autoradiography. Autoradiography revealed a higher cortex-to-cerebellum ratio in the transgenic mice than in the controls. Results from autoradiography were validated by immunohistochemistry and poly-GA immunoassays. Moreover, we confirmed antibody uptake in the CNS in a pharmacokinetic study of the perfused tissues. In summary, [64Cu]Cu-NODAGA-mAb1A12 demonstrated favorable in vitro characteristics and an increased relative binding in poly-GA transgenic mice compared to wild-type mice in vivo. Our results with this first-in-class radiotracer suggested that targeting poly-GA is a promising approach for PET/CT imaging in FTD/ALS.

TDP-43 pathology in subacute sclerosing panencephalitis.

Subacute sclerosing panencephalitis (SSPE) is a fatal, slowly progressive brain disorder caused by a mutated measles virus. Both subacute inflammatory and neurodegenerative mechanisms appear to play significant roles in the pathogenesis. TAR DNA-binding protein 43 (TDP-43) inclusions are a common co-pathology in several neurodegenerative disorders with diverse pathogenesis. In the present study, we examined brains of 16 autopsied SSPE patients for the presence of TDP-43 pathology and possible associations with tau pathology. Immunohistochemical staining identified TDP-43 inclusions in 31% of SSPE cases. TDP-43 pathology was widely distributed in the brains, most severely in the atrophied cerebral cortex (temporal and parietal), and most frequently as tangle- and thread-like neuronal cytoplasmic inclusions. It was associated with longer disease duration (>4 years) and tau pathology (all TDP-43-positive cases had tau-positive neurofibrillary tangles). This study demonstrates for the first time an association between TDP-43 pathology and SSPE. The co-occurrence of TDP-43 and tau aggregates and correlation with the disease duration suggest that both pathological proteins are involved in the neurodegenerative process induced by viral inflammation.

TDP-43 and Alzheimer's Disease Pathology in the Brain of a Harbor Porpoise Exposed to the Cyanobacterial Toxin BMAA.

Cetaceans are well-regarded as sentinels for toxin exposure. Emerging studies suggest that cetaceans can also develop neuropathological changes associated with neurodegenerative disease. The occurrence of neuropathology makes cetaceans an ideal species for examining the impact of marine toxins on the brain across the lifespan. Here, we describe TAR DNA-binding protein 43 (TDP-43) proteinopathy and Alzheimer's disease (AD) neuropathological changes in a beached harbor porpoise (Phocoena phocoena) that was exposed to a toxin produced by cyanobacteria called β-N-methylamino-L-alanine (BMAA). We found pathogenic TDP-43 cytoplasmic inclusions in neurons throughout the cerebral cortex, midbrain and brainstem. P62/sequestosome-1, responsible for the autophagy of misfolded proteins, was observed in the amygdala, hippocampus and frontal cortex. Genes implicated in AD and TDP-43 neuropathology such as APP and TARDBP were expressed in the brain. AD neuropathological changes such as amyloid-β plaques, neurofibrillary tangles, granulovacuolar degeneration and Hirano bodies were present in the hippocampus. These findings further support the development of progressive neurodegenerative disease in cetaceans and a potential causative link to cyanobacterial toxins. Climate change, nutrient pollution and industrial waste are increasing the frequency of harmful cyanobacterial blooms. Cyanotoxins like BMAA that are associated with neurodegenerative disease pose an increasing public health risk.

Selective vulnerability of subcortical nuclei in atypical Alzheimer’s Disease

AbstractBackgroundRecent evidence suggests that sleep‐wake dysregulation in Alzheimer’s disease (AD) is an early event and parallels the tau accumulation in the arousal system spefically noted in the locus coeruleus (LC) as well as other subcortical nuclei as the Pedunculopontine Tegmental Nucleus (PPTg), Laterodorsal Tegmental Nucleus (LDTg), and the Dorsal Raphe (DR). We have previously demonstrated that the pattern of sleep‐wake dysfunction is different among AD clinical variants. This existence of specific sleep‐wake profiles suggest differential degeneration patterns of the arousal system within differing AD presentations. This study continues this investigation at the cellular/molecular level to uncover the selective vulnerability of subcortical structures to AD specific tau accumulation. That remains elusive to‐date.MethodHuman postmortem brainstem samples were sourced from the Neurodegenerative Disease Brain Bank (NDBB) at UCSF. 24 subjects were selected based on primary diagnosis, including 6 amnestic AD, 17 non‐amnestic AD (5 EOAD, 3 PCA, 5 lvPPA, 4 CBS) and 1 control. Paraffin blocks containing the areas of interest were cut into 8‐lm‐thick sections that underwent double‐labeled immunohistochemistry (IHC) and brightfield imaging. The images were then overlayed to created mutlidimentaional maps and neurons were classified in 4 subgroups: (1) positive for the neurotransmitter of interest (NEU‐positive), (2) bearing hyperphosphorylated tau neuronal cytoplasmic inclusions (tau‐positive), (3) positive for the neurotransmitter of interest and bearing tau‐positive (NEU‐positive/tau‐positive colocalized neurons), and (4) negative for NEU and tau.ResultIn the LC, lvPPA had the highest had the highest percentage of tau postive neurons (40%), followed by amnestic AD (31.1%), PCA (29.3%), EOAD (27.1%), and finally CBS (24.3%). EOAD had the highest percentage of colocalized neurons (34.7%) as well as LOAD (31%) compared to PCA (0.02%), lvPPA (0.18%), CBS (0.09%).ConclusionMultidimensional quantitative analysis suggests differences in vulnerabliblity of sleep‐wake subcortical nuclei to AD, EOAD, PCA, lvPPA, and CBS. For instance the neuronal loss in EOAD was so extreme in the LC that few neurons remained to develop aggregates whereas in PCA, CBS, and lvPPA the high percentage of tau accumulation presents a neuropathological framework underlying atypical clinical manifestations and possible insight into therapeutic targets.

Heterogeneity of cortical pTDP-43 inclusion morphologies in amyotrophic lateral sclerosis

BackgroundDespite the presence of significant cortical pTDP-43 inclusions of heterogeneous morphologies in patients diagnosed with amyotrophic lateral sclerosis (ALS), pathological subclassification is routinely performed in the minority of patients with concomitant frontotemporal dementia (FTD).ObjectiveIn order to improve current understanding of the presence and relevance of pathological pTDP-43 subtypes in ALS, the present study examined the pattern of cortical pTDP-43 aggregates in 61 ALS cases without FTD.ResultsBased on the presence, morphology and composition of pTDP-43 pathology, three distinct ALS-TDP subtypes were delineated: (1) A predominant pattern of pTDP-43 granulofilamentous neuronal inclusions (GFNIs) and grains that were immuno-negative for p62 was identified in 18% of cases designated ALS-TDP type E; (2) neuronal cytoplasmic inclusions (NCIs) that were immuno-positive for both pTDP-43 and p62 were observed in 67% of cases assigned ALS-TDP type B; and (3) scarce cortical pTDP-43 and p62 aggregates were identified in 15% of cases coined ALS-TDP type SC (scarce cortical). Quantitative analyses revealed a significantly greater burden of pTDP-43 GFNI and grains in ALS-TDP type E. Principal component analysis demonstrated significant relationships between GFNIs, grains and ALS-TDP subtypes to support the distinction of subtypes E and B. No significant difference in age at death or disease duration was found between ALS-TDP subgroups to suggest that these subtypes represent earlier or later stages of the same disease process. Instead, a significantly higher ALS-TDP stage, indicating greater topographical spread of pTDP-43, was identified in ALS-TDP type E. Alzheimer’s disease neuropathological change (ABC score ≥ intermediate) and Lewy body disease (Braak stage ≥ IV) was more prevalent in the ALS-TDP type SC cohort, which also demonstrated a significantly lower overall cognitive score.ConclusionIn summary, the present study demonstrates that ALS-TDP does not represent a single homogenous neuropathology. We propose the subclassification of ALS-TDP into three distinct subtypes using standard immuno-stains for pTDP-43 and p62 in the motor cortex, which is routinely sampled and evaluated for diagnostic neuropathological characterisation of ALS. We propose that future studies specify both clinicopathological group and pTDP-43 subtype to advance current understanding of the pathogenesis of clinical phenotypes in pTDP-43 proteinopathies, which will have significant relevance to the development of targeted therapies for this heterogeneous disorder.

Neuronal models of TDP-43 proteinopathy display reduced axonal translation, increased oxidative stress, and defective exocytosis.

Amyotrophic lateral sclerosis (ALS) is a progressive, lethal neurodegenerative disease mostly affecting people around 50-60 years of age. TDP-43, an RNA-binding protein involved in pre-mRNA splicing and controlling mRNA stability and translation, forms neuronal cytoplasmic inclusions in an overwhelming majority of ALS patients, a phenomenon referred to as TDP-43 proteinopathy. These cytoplasmic aggregates disrupt mRNA transport and localization. The axon, like dendrites, is a site of mRNA translation, permitting the local synthesis of selected proteins. This is especially relevant in upper and lower motor neurons, whose axon spans long distances, likely accentuating their susceptibility to ALS-related noxae. In this work we have generated and characterized two cellular models, consisting of virtually pure populations of primary mouse cortical neurons expressing a human TDP-43 fusion protein, wt or carrying an ALS mutation. Both forms facilitate cytoplasmic aggregate formation, unlike the corresponding native proteins, giving rise to bona fide primary culture models of TDP-43 proteinopathy. Neurons expressing TDP-43 fusion proteins exhibit a global impairment in axonal protein synthesis, an increase in oxidative stress, and defects in presynaptic function and electrical activity. These changes correlate with deregulation of axonal levels of polysome-engaged mRNAs playing relevant roles in the same processes. Our data support the emerging notion that deregulation of mRNA metabolism and of axonal mRNA transport may trigger the dying-back neuropathy that initiates motor neuron degeneration in ALS.

Increased expression of human antiviral protein MxA in FUS proteinopathy in amyotrophic lateral sclerosis.

FUS mutations are one of the major mutations in familial amyotrophic lateral sclerosis (ALS). The pathological hallmark is FUS-positive neuronal cytoplasmic inclusions (FUS-NCI), known as FUS proteinopathy. Human myxovirus resistance protein 1 (MxA) is an IFN-induced dynamin-like GTPase that acts as antiviral factor. In this study, we examined the expression of MxA in neurons bearing FUS-NCI. We performed immunohistochemistry for FUS and MxA to examine the expression of MxA in two autopsy cases with different FUS gene mutations localized at the nuclear localization signal site (Case 1, H517P; Case 2, R521C). MxA. Most neurons bearing FUS-NCI have increased cytoplasmic MxA expression. Increased cytoplasmic MxA showed several distribution patterns in relation to FUS-NCIs such as the following: colocalization with NCI, distribution more widely than NCI, and different distribution peaks from NCI. Our results suggested that antiviral signaling IFNs are involved upstream in the formation of FUS-NCI in ALS-FUS patients.

Lewy body disease as a potential negative outcome modifier of glioblastoma treatment: a case report

BackgroundElderly patients with glioblastoma are particularly susceptible to the adverse effects of ionizing radiation to the brain. This population also has an increasing prevalence of dementia in the successive seventh, eighth and nineth decade of life, and dementia with Lewy bodies is characterized by pathologic α-synucleins, proteins that take part in neuronal DNA damage repair.Case presentationWe report a 77-year-old man, with a history of coronary artery disease and mild cognitive impairment, who experienced subacute behavioral changes over 3 months with wording-finding difficulty, memory loss, confusion, perseveration, and irritable mood. Neuroimaging studies disclosed a 2.5 × 2.4 × 2.7 cm cystic enhancing mass with central necrosis in the left temporal lobe of the brain. Gross total resection of the tumor revealed IDH-1 wild-type glioblastoma. After treatment with radiation and temozolomide chemotherapy, his cognitive status deteriorated rapidly, and he died from unexpected sudden death 2 months after radiation. Autopsy of his brain revealed (i) tumor cells with atypical nuclei and small lymphocytes, (ii) neuronal cytoplasmic inclusions and Lewy bodies that were positive for α-synuclein in the midbrain, pons, amygdala, putamen and globus pallidus, and (iii) no amyloid plaques and only rare neurofibrillary tangles near the hippocampi.ConclusionsThis patient most likely had pre-clinical limbic subtype of dementia with Lewy bodies prior to his diagnosis of glioblastoma. The radiation and temozolomide that was used to treat his tumor may have accelerated neuronal damage due to induction of DNA breakage when his brain was already compromised by pathologic α-synucleins. α-Synucleinopathy could be a negative outcome modifier in glioblastoma patients.

Structural polymorphism of the low-complexity C-terminal domain of TDP-43 amyloid aggregates revealed by solid-state NMR.

Aberrant aggregation of the transactive response DNA-binding protein (TDP-43) is associated with several lethal neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal dementia. Cytoplasmic neuronal inclusions of TDP-43 are enriched in various fragments of the low-complexity C-terminal domain and are associated with different neurotoxicity. Here we dissect the structural basis of TDP-43 polymorphism using magic-angle spinning solid-state NMR spectroscopy in combination with electron microscopy and Fourier-transform infrared spectroscopy. We demonstrate that various low-complexity C-terminal fragments, namely TDP-13 (TDP-43300-414), TDP-11 (TDP-43300-399), and TDP-10 (TDP-43314-414), adopt distinct polymorphic structures in their amyloid fibrillar state. Our work demonstrates that the removal of less than 10% of the low-complexity sequence at N- and C-termini generates amyloid fibrils with comparable macroscopic features but different local structural arrangement. It highlights that the assembly mechanism of TDP-43, in addition to the aggregation of the hydrophobic region, is also driven by complex interactions involving low-complexity aggregation-prone segments that are a potential source of structural polymorphism.

Clusters of co-abundant proteins in the brain cortex associated with fronto-temporal lobar degeneration

BackgroundFrontotemporal lobar degeneration (FTLD) is characterized pathologically by neuronal and glial inclusions of hyperphosphorylated tau or by neuronal cytoplasmic inclusions of TDP43. This study aimed at deciphering the molecular mechanisms leading to these distinct pathological subtypes.MethodsTo this end, we performed an unbiased mass spectrometry-based proteomic and systems-level analysis of the middle frontal gyrus cortices of FTLD-tau (n = 6), FTLD-TDP (n = 15), and control patients (n = 5). We validated these results in an independent patient cohort (total n = 24).ResultsThe middle frontal gyrus cortex proteome was most significantly altered in FTLD-tau compared to controls (294 differentially expressed proteins at FDR = 0.05). The proteomic modifications in FTLD-TDP were more heterogeneous (49 differentially expressed proteins at FDR = 0.1). Weighted co-expression network analysis revealed 17 modules of co-regulated proteins, 13 of which were dysregulated in FTLD-tau. These modules included proteins associated with oxidative phosphorylation, scavenger mechanisms, chromatin regulation, and clathrin-mediated transport in both the frontal and temporal cortex of FTLD-tau. The most strongly dysregulated subnetworks identified cyclin-dependent kinase 5 (CDK5) and polypyrimidine tract-binding protein 1 (PTBP1) as key players in the disease process. Dysregulation of 9 of these modules was confirmed in independent validation data sets of FLTD-tau and control temporal and frontal cortex (total n = 24). Dysregulated modules were primarily associated with changes in astrocyte and endothelial cell protein abundance levels, indicating pathological changes in FTD are not limited to neurons.ConclusionsUsing this innovative workflow and zooming in on the most strongly dysregulated proteins of the identified modules, we were able to identify disease-associated mechanisms in FTLD-tau with high potential as biomarkers and/or therapeutic targets.

Differential immunophenotypes of neuronal cytoplasmic inclusions in the dentate gyrus of multiple system atrophy and their association with clinicopathological features.

Although hippocampal pathologies of multiple system atrophy (MSA) and their association with dementia have been reported, no studies have reported clinicopathological differences among MSA patients with and without neuronal cytoplasmic inclusions (NCIs) in the dentate gyrus (dntNCIs). We investigated hippocampal NCI pathology in 18 MSA patient autopsies, focusing on phosphorylated α-synuclein (pAS)- and phosphorylated tau (pT)-positive dntNCIs. There were 8 MSA patients without and 10 with dntNCIs. The latter group was subclassified by immunophenotype: those with pAS-positive dntNCIs (pAS-dntNCI subtype), those with pT-positive dntNCIs (pT-dntNCI subtype), and those with both types of dntNCIs. MSA patients with dntNCIs survived longer with prolonged tracheostomy and had dementia more frequently than those without dntNCIs. The brain weights of patients with dntNCIs were lower than those without dntNCIs. The presence of dementia was similar among the dntNCI subtypes. The pAS-dntNCI subtype was associated with longer survival and smaller brain weights; the pT-dntNCI subtype exhibited more frequent tau pathologies than the pAS-dntNCI subtype. Thus, MSA with dntNCIs is a possible pathological subtype of longer survivors that correlates with longer disease duration, prolonged tracheostomy, and high frequency of dementia. Understanding clinicopathological differences in MSA patients with and without dntNCIs may lead to improved personalized management strategies.

Lessons learned from a sporadic FUSopathy in a young man: a case report

BackgroundIn frontotemporal dementia (FTD) spectrum, younger patients may correspond to fusopathy cases, and cognitive decline could be rapidly progressive. We present a clinical and neuropathological description of a patient.Case presentationA 37-year-old man, without a family history of neurodegenerative diseases, was brought by his family to consult for dysarthria and behavioural change. Initial exploration showed spastic dysarthria and disinhibition. He progressively worsened with a pseudobulbar syndrome, right-lateralized pyramidal signs, left hemispheric corticobasal syndrome and, finally, lower motor neuron signs in his right arm. He died four years after the initiation of the syndrome from bronchopneumonia. Laboratory tests (including blood and cerebrospinal fluid (CSF)) were normal. Magnetic resonance imaging (MRI) and fluorodeoxyglucose-containing positron emission tomography (PET-18F-FDG) showed left fronto-insular atrophy and hypometabolism. Subsequently, 123I-ioflupane (DaT-SCAN®) single-photon emission computed tomography (SPECT) was pathologic, manifesting bilaterally decreased activity with greater affection on the left side. Only a third electromyogram (EMG) detected denervation in the last year of evolution. No mutations were found in genes such as Tau, progranulin, C9orf72, FUS, TDP-43, CHMP2B, or VCP. In necropsy, severe frontotemporal atrophy with basophilic neuronal cytoplasmic and intranuclear inclusions, negative for tau and TAR DNA binding protein 43 (TDP-43), but positive for fused in sarcoma (FUS) consistent with specifically basophilic inclusions body disease (BIBD) type was found.ConclusionsIn patients affected by FTD, particularly the youngest, with rapidly progressive decline and early motor affection, fusopathy must be suspected. These cases can include motor signs described in the FTD spectrum. Lower motor neuron affection in EMG could be detected late.

Mutated FUS in familial amyotrophic lateral sclerosis involves multiple hnRNPs in the formation of neuronal cytoplasmic inclusions.

Fused in sarcoma (FUS), coded by FUS, is a heterogeneous nuclear ribonucleoprotein (hnRNP). FUS mutations are among the major mutations in familial amyotrophic lateral sclerosis (ALS-FUS: ALS6). The pathological hallmarks of ALS-FUS are FUS-positive neuronal cytoplasmic inclusions (NCI). We examined various hnRNPs in FUS NCIs in the hippocampus in ALS-FUS cases with different FUS mutations (Case 1, H517P; Case 2, R521C). We also examined TDP43-positive NCIs in sporadic ALS hippocampi. Immunohistochemistry was performed using primary antibodies against FUS, p-TDP43, TDP43, hnRNPA1, hnRNPD, PCBP1, PCBP2, and p62. Numerous FUS inclusions were found in the hippocampal granule and pyramidal cell layers. Double immunofluorescence revealed colocalization of FUS and p-TDP43, and FUS and PCBP2 (p-TDP43/FUS: 64.3%, PCBP2/FUS: 23.9%). Colocalization of FUS and PCBP1, however, was rare (PCBP1/FUS: 7.6%). In the hippocampi of patients with sporadic ALS, no colocalization was observed between TDP43-positive inclusions and other hnRNPs. This is the first study to show that FUS inclusions colocalize with other hnRNPs, such as TDP43, PCBP2, and PCBP1. These findings suggest that in ALS-FUS, FUS inclusions are the initiators, followed by alterations of multiple other hnRNPs, resulting in impaired RNA metabolism.

Molecular fragment characteristics and distribution of tangle associated TDP-43 (TATs) and other TDP-43 lesions in Alzheimer's disease.

TAR DNA binding protein 43 (TDP-43) pathology is a defining feature of frontotemporal lobar degeneration (FTLD). In FTLD-TDP there is a moderate-to-high burden of morphologically distinctive TDP-43 immunoreactive inclusions distributed throughout the brain. In Alzheimer's disease (AD), similar TDP-43 immunoreactive inclusions are observed. In AD, however, there is a unique phenomenon of neurofibrillary tangle-associated TDP-43 (TATs) whereby TDP-43 intermingles with neurofibrillary tangles. Little is known about the characteristics and distribution of TATs, or how burden and distribution of TATs compares to burden and distribution of other FTLD-TDP-like lesions observed in AD. Here we characterize molecular fragment characteristics, burden and distribution of TATs and assess how these features compare to features of other TDP-43 lesions. We performed TDP-43 immunohistochemistry with anti-phosphorylated, C- and N-terminal TDP-43 antibodies in 20 high-probability AD cases and semi-quantitative burden of seven inclusion types within five brain regions (entorhinal cortex, subiculum, CA1 and dentate gyrus of hippocampus, occipitotemporal cortex). Hierarchical cluster analysis was used to analyze the dataset that consisted of 75 different combinations of neuropathological features. TATs were nonspherical with heterogeneous staining patterns and present in all regions except hippocampal dentate. All three antibodies detected TATs although N-terminal antibody sensitivity was low. Three clusters were identified: Cluster-1 had mild-moderate TATs, moderate-frequent neuronal cytoplasmic inclusions, dystrophic neurites, neuronal intranuclear inclusions and fine neurites, and perivascular and granular inclusions identified only with the N-terminal antibody throughout the brain; Cluster-2 had scant TATs in limbic regions and Cluster-3 mild-moderate TATs and mild-moderate neuronal cytoplasmic inclusions and dystrophic neurites throughout the brain and moderate fine neurites. Only 17% of cluster 1 cases had the TMEM106b GG (protective) haplotype and 83% had hippocampal sclerosis. Both features differed across clusters (p=0.03 & p=0.01). TATs have molecular characteristics, distribution and burden, and genetic and pathologic associations like FTLD-TDP lesions.

PARP1 Activation Controls Stress Granule Assembly after Oxidative Stress and DNA Damage

DNA damage causes PARP1 activation in the nucleus to set up the machinery responsible for the DNA damage response. Here, we report that, in contrast to cytoplasmic PARPs, the synthesis of poly(ADP-ribose) by PARP1 opposes the formation of cytoplasmic mRNA-rich granules after arsenite exposure by reducing polysome dissociation. However, when mRNA-rich granules are pre-formed, whether in the cytoplasm or nucleus, PARP1 activation positively regulates their assembly, though without additional recruitment of poly(ADP-ribose) in stress granules. In addition, PARP1 promotes the formation of TDP-43- and FUS-rich granules in the cytoplasm, two RNA-binding proteins which form neuronal cytoplasmic inclusions observed in certain neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Together, the results therefore reveal a dual role of PARP1 activation which, on the one hand, prevents the early stage of stress granule assembly and, on the other hand, enables the persistence of cytoplasmic mRNA-rich granules in cells which may be detrimental in aging neurons.

Widespread CNS pathology in amyotrophic lateral sclerosis homozygous for the D90A SOD1 mutation

Mutations in the gene encoding the ubiquitously expressed free radical scavenging enzyme superoxide dismutase-1 (SOD1) are found in 2–6% of amyotrophic lateral sclerosis patients. The most frequent SOD1 mutation worldwide is D90A. Amyotrophic lateral sclerosis caused by this mutation has some unusual features: the heredity is usually recessive, the phenotype is stereotypic with slowly evolving motor symptoms beginning in the legs and may also include sensory, autonomic, and urinary bladder involvement. Furthermore, the mutant protein resembles the wild type, with normal content and enzymatic activity in the central nervous system. Here, we report neuropathological findings in nine patients homozygous for the D90A mutation. All nine had numerous small granular inclusions immunoreactive for misfolded SOD1 in motor neurons and glial nuclei in the spinal cord and brainstem. In addition to degeneration of the corticospinal tracts, all patients had degeneration of the dorsal columns. We also found intense gliosis in circumscribed cortical areas of the frontal and temporal lobes and in the insula. In these areas and in adjacent white matter, there were SOD1 staining neuropil threads. A few SOD1-immunopositive cytoplasmic neuronal inclusions were observed in cortical areas, as were glial nuclear inclusions. As suggested by the symptoms and signs and earlier neurophysiological and imaging investigations, the histopathology in patients homozygous for the D90A SOD1 extends beyond the motor system to include cognitive and sensory cortical areas. However, even in the patients that had a symptomatic disease duration of more than 2 or 3 decades and lived into their 70s or 80s, there were no SOD1-inclusion pathology and no typical dysfunction (apart from the musculature) in non-nervous organs. Thus, only specific parts of the CNS seem to be vulnerable to toxicity provoked by homozygously expressed mutant SOD1.