Single-nucleus transcriptome atlas of orbitofrontal cortex in amyotrophic lateral sclerosis with a deep learning-based decoding of alternative polyadenylation mechanisms.

Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are two age-related and fatal neurodegenerative disorders that lie on a shared disease spectrum. While both disorders involve complex interactions between neuronal and glial cells, the specific cell-type alterations and their contributions to disease pathophysiology remain incompletely understood. Here, we applied single-nucleus RNA sequencing of the orbitofrontal cortex, a region affected in ALS-FTLD, to map cell-type specific transcriptional signatures in C9orf72-related ALS (with and without FTLD) and sporadic ALS cases.

A C-Terminally Truncated TDP-43 Splice Isoform Exhibits Neuronal Specific Cytoplasmic Aggregation and Contributes to TDP-43 Pathology in ALS.

Neuronal cytoplasmic aggregation and ubiquitination of TDP-43 is the most common disease pathology linking Amyotrophic Lateral Sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). TDP-43 pathology is characterized by the presence of low molecular weight TDP-43 species generated through proteolytic cleavage and/or abnormal RNA processing events. In addition to N-terminally truncated TDP-43 species, it has become evident that C-terminally truncated variants generated through alternative splicing in exon 6 also contribute to the pathophysiology of ALS/FTLD.

TDP-43 stabilizes G3BP1 mRNA: relevance to amyotrophic lateral sclerosis/frontotemporal dementia.

TDP-43 nuclear depletion and concurrent cytoplasmic accumulation in vulnerable neurons is a hallmark feature of progressive neurodegenerative proteinopathies such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Cellular stress signalling and stress granule dynamics are now recognized to play a role in ALS/FTD pathogenesis. Defective stress granule assembly is associated with increased cellular vulnerability and death.

Synaptic localization of C9orf72 regulates post-synaptic glutamate receptor 1 levels.

A hexanucleotide repeat expansion in a noncoding region of C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Reduction of select or total C9orf72 transcript and protein levels is observed in postmortem C9-ALS/FTD tissue, and loss of C9orf72 orthologues in zebrafish and C. elegans results in motor deficits.

Unaffected mosaic case: RNA foci, dipeptide proteins, but upregulated C9orf72 expression.

Objective: Suggested disease mechanisms for amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration include haploinsufficiency, GC/CG RNA foci, and dipeptide repeat (DPR) proteins translated from the GC expansion; however, the role of small expansions (e.g., 30-90 repeats) is unknown and was investigated here.

Immunohistochemical detection of C9orf72 protein in frontotemporal lobar degeneration and motor neurone disease: patterns of immunostaining and an evaluation of commercial antibodies.

We have employed as 'gold standards' two in-house, well-characterised and validated polyclonal antibodies, C9-L and C9-S, which detect the longer and shorter forms of C9orf72, and have compared seven other commercially available antibodies with these in order to evaluate the utility of the latter as credible tools for the demonstration of C9orf72. C9-L and C9-S antibodies immunostained cytoplasmic 'speckles', and the nuclear membrane, respectively, in cerebellar Purkinje cells of the cerebellum in patients with behavioural variant frontotemporal dementia (bvFTD) with amyotrophic lateral sclerosis (ALS), and in patients with ALS alone. Similar staining was seen in Purkinje cells in healthy control tissues and in other neurodegenerative disorders, and in pyramidal cells of CA4 and dentate gyrus of hippocampus.

C9orf72 isoforms in Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Degeneration.

A hexanucleotide (G4C2) repeat expansion in the 5' non-coding region C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Three modes of toxicity have been proposed: gain of function through formation of RNA foci and sequestration of RNA binding proteins; expression of dipeptide repeat proteins generated by repeat-associated non-ATG translation; and loss of function due to C9orf72 haploinsufficiency. Much is known about the proposed gain of function mechanisms, but there is little knowledge of the normal function of C9orf72 and the cellular consequences if its activity is perturbed.

MTHFSD and DDX58 are novel RNA-binding proteins abnormally regulated in amyotrophic lateral sclerosis.

Tar DNA-binding protein 43 (TDP-43) is an RNA-binding protein normally localized to the nucleus of cells, where it elicits functions related to RNA metabolism such as transcriptional regulation and alternative splicing. In amyotrophic lateral sclerosis, TDP-43 is mislocalized from the nucleus to the cytoplasm of diseased motor neurons, forming ubiquitinated inclusions. Although mutations in the gene encoding TDP-43, TARDBP, are found in amyotrophic lateral sclerosis, these are rare.

Isoform-specific antibodies reveal distinct subcellular localizations of C9orf72 in amyotrophic lateral sclerosis.

Objective: A noncoding hexanucleotide repeat expansion in C9orf72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). It has been reported that the repeat expansion causes a downregulation of C9orf72 transcripts, suggesting that haploinsufficiency may contribute to disease pathogenesis. Two protein isoforms are generated from three alternatively spliced transcripts of C9orf72; a long form (C9-L) and a short form (C9-S), and their function(s) are largely unknown owing to lack of specific antibodies.

Low molecular weight species of TDP-43 generated by abnormal splicing form inclusions in amyotrophic lateral sclerosis and result in motor neuron death.

The presence of lower molecular weight species comprising the C-terminal region of TAR DNA-binding protein 43 (TDP-43) is a characteristic of TDP-43 proteinopathy in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Here, we have identified a novel splice variant of TDP-43 that is upregulated in ALS and generates a 35-kDa N-terminally truncated species through use of an alternate translation initiation codon (ATG(Met85)), denoted here as Met(85)-TDP-35. Met(85)-TDP-35 expressed ectopically in human neuroblastoma cells exhibited reduced solubility, cytoplasmic distribution, and aggregation.

A two-hybrid screen identifies an unconventional role for the intermediate filament peripherin in regulating the subcellular distribution of the SNAP25-interacting protein, SIP30.

Peripherin is a type III intermediate filament protein, the expression of which is associated with the acquisition and maintenance of a terminally differentiated neuronal phenotype. Peripherin up-regulation occurs during acute neuronal injury and in degenerating motor neurons of amyotrophic lateral sclerosis. The functional role(s) of peripherin during normal, injurious, and disease conditions remains unknown, but may be related to differential expression of spliced isoforms.

Gene targeting of mouse Tardbp negatively affects Masp2 expression.

Amyotrophic Lateral Sclerosis (ALS) is a devastating adult onset neurodegenerative disease affecting both upper and lower motor neurons. TDP-43, encoded by the TARDBP gene, was identified as a component of motor neuron cytoplasmic inclusions in both familial and sporadic ALS and has become a pathological signature of the disease. TDP-43 is a nuclear protein involved in RNA metabolism, however in ALS, TDP-43 is mislocalized to the cytoplasm of affected motor neurons, suggesting that disease might be caused by TDP-43 loss of function.

Genome-wide association analyses in Han Chinese identify two new susceptibility loci for amyotrophic lateral sclerosis.

To identify susceptibility genes for amyotrophic lateral sclerosis (ALS), we conducted a genome-wide association study (GWAS) in 506 individuals with sporadic ALS and 1,859 controls of Han Chinese ancestry. Ninety top SNPs suggested by the current GWAS and 6 SNPs identified by previous GWAS were analyzed in an independent cohort of 706 individuals with ALS and 1,777 controls of Han Chinese ancestry. We discovered two new susceptibility loci for ALS at 1q32 (CAMK1G, rs6703183, Pcombined = 2.

RNA targets of TDP-43 identified by UV-CLIP are deregulated in ALS.

TDP-43 is a predominantly nuclear DNA/RNA binding protein involved in transcriptional regulation and RNA processing. TDP-43 is also a component of the cytoplasmic inclusion bodies characteristic of amyotrophic lateral sclerosis (ALS) and of frontotemporal lobar degeneration with ubiquitinated inclusions (FTLD-U). We have investigated the premise that abnormalities of TDP-43 in disease would be reflected by changes in processing of its target RNAs.

An aggregate-inducing peripherin isoform generated through intron retention is upregulated in amyotrophic lateral sclerosis and associated with disease pathology.

The neuronal intermediate filament protein peripherin is a component of ubiquitinated inclusions and of axonal spheroids in amyotrophic lateral sclerosis (ALS). Overexpression of peripherin causes motor neuron degeneration in transgenic mice and variations within the peripherin gene have been identified in ALS cases. We have shown previously the abnormal expression of a neurotoxic peripherin splice variant in transgenic mice expressing mutant superoxide dismutase-1.

A novel peripherin isoform generated by alternative translation is required for normal filament network formation.

Peripherin is a type III neuronal intermediate filament protein detected within the intraneuronal inclusions characteristic of amyotrophic lateral sclerosis. The constitutively expressed peripherin isoform is encoded by all nine exons of the human and mouse peripherin genes to generate a protein species of approximately 58 kDa on sodium dodecyl sulfate-polyacrylamide gels. Expression of this isoform, termed Per-58, generates a filament network in transfected SW13 vim cells.

Evidence that TDP-43 is not the major ubiquitinated target within the pathological inclusions of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disease characterized by the presence of various types of ubiquitinated inclusions in the cytoplasm of affected motor neurons. The identification of the ubiquitinated targets within these inclusions has represented a major challenge, as this may provide new gene candidates and/or clues to understanding the neurodegenerative mechanism(s) underlying the disease. As such, the nuclear factor TAR DNA-binding protein (TDP-43) was recently identified as a component of ubiquitinated skein-like inclusions and round inclusions in ALS.

Lack of TDP-43 abnormalities in mutant SOD1 transgenic mice shows disparity with ALS.

Mislocalization of the TAR-DNA binding protein (TDP-43) from the nucleus to the cytoplasm of diseased motor neurons and association with intraneuronal ubiquitinated inclusions has recently been reported in amyotrophic lateral sclerosis (ALS). Here, we have investigated TDP-43 immunoreactivity in three lines of mutant SOD1 transgenic mice, G93A, G37R and G85R and compared with labeling in one sporadic ALS case and two familial ALS cases carrying mutations in SOD1, A4T and I113T. Our findings show that there is no mislocalization of TDP-43 to the cytoplasm in motor neurons of mutant SOD1 transgenic mice, nor association of TDP-43 with ubiquitinated inclusions.

Genetic studies of GRN and IFT74 in amyotrophic lateral sclerosis.

There is increasing evidence of a clinical, neuropathological and genetic overlap between frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). We conducted a case-control study using a UK dataset to test the hypothesis that polymorphisms in two FTD-related genes (GRN and FT74) are associated with increased susceptibility to ALS. We evaluated the majority of known genetic variability in IFT74 and GRN.

Neuronal intermediate filaments and ALS: a new look at an old question.

One of the pathological hallmarks of ALS is the presence of axonal spheroids and perikaryal accumulations/aggregations comprised of the neuronal intermediate filament proteins, neurofilaments and peripherin. These abnormalities represent a point of convergence of both familial and sporadic forms of the disease and understanding their formation may reveal shared pathways in what is otherwise considered a highly heterogeneous disorder. Here we provide a review of the basic biology of neurofilaments and peripherin and the evidence linking them with ALS disease pathogenesis.

A study of the ultrastructure and gene location of hereditary gingival fibromatosis.

Objective: To ascertain histology changes of hereditary gingival fibromatosis (HGF) and the location of HGF gene.

Methods: A pedigree analyses of HGF; and the ultrastructure of gingival overgrowth tissue was observed with electron microscopy. The overgrowth of the HGF gene was defined with microsatellite markers.

The gamma S-crystallin gene is mutated in autosomal recessive cataract in mouse.

We established a recessive cataract model from a spontaneous mutation in the KUNMING outbred mice. Lens opacity appears 11 days after birth. Slit lamp examination reveals that the opacity mainly localizes to the nuclear region of the lens.