Oligogenic inheritance of optineurin (OPTN) and C9ORF72 mutations in ALS highlights localisation of OPTN in the TDP‐43‐negative inclusions of C9ORF72‐ALS

Amyotrophic lateral sclerosis (ALS) is characterized by motor neurone loss resulting in muscle weakness, spasticity and ultimately death. 5‐10% are caused by inherited mutations, most commonly C9ORF72, SOD1, TARDBP and FUS. Rarer genetic causes of ALS include mutation of optineurin (mt OPTN). Furthermore, optineurin protein has been localized to the ubiquitylated aggregates in several neurodegenerative diseases, including ALS. This study: (i) investigated the frequency of mt OPTN in ALS patients in England; (ii) characterized the clinical and neuropathological features of ALS associated with a mt OPTN; and (iii) investigated optineurin neuropathology in C9ORF72‐related ALS (C9ORF72‐ALS). We identified a heterozygous p.E322K missense mutation in exon 10 of OPTN in one familial ALS patient who additionally had a C9ORF72 mutation. This patient had bulbar, limb and respiratory disease without cognitive problems. Neuropathology revealed motor neurone loss, trans‐activation response DNA protein 43 (TDP‐43)‐positive neuronal and glial cytoplasmic inclusions together with TDP‐43‐negative neuronal cytoplasmic inclusions in extra motor regions that are characteristic of C9ORF72‐ALS. We have demonstrated that both TDP‐43‐positive and negative inclusion types had positive staining for optineurin by immunohistochemistry. We went on to show that optineurin was present in TDP‐43‐negative cytoplasmic extra motor inclusions in C9ORF72‐ALS cases that do not carry mt OPTN. We conclude that: (i) OPTN mutations are associated with ALS; (ii) optineurin protein is present in a subset of the extramotor inclusions of C9ORF72‐ALS; (iii) It is not uncommon for multiple ALS‐causing mutations to occur in the same patient; and (iv) studies of optineurin are likely to provide useful dataregarding the pathophysiology of ALS and neurodegeneration.     

Frontotemporal lobar degeneration with TDP‐43 proteinopathy and chromosome 9p repeat expansion in C9ORF72: clinicopathologic correlation

Mutations in C9ORF72 resulting in expanded hexanucleotide repeats were recently reported to be the underlying genetic abnormality in chromosome 9p‐linked frontotemporal lobar degeneration with TAR DNA‐binding protein of 43 kD (TDP‐43) proteinopathy (FTLD‐TDP), amyotrophic lateral sclerosis (ALS), and frontotemporal lobar degeneration with motor neuron disease (FTLD‐MND). Several subsequent publications described the neuropathology as being similar to that of FTLD‐TDP and ALS without C9ORF72 mutations, except that cases with mutations have p62 and ubiquitin positive, TDP‐43 negative inclusions in cerebellum, hippocampus, neocortex, and basal ganglia. The identity of this protein is as yet unknown, and its significance is unclear. With the goal of potentially uncovering the significance of these inclusions, we compared the clinical, pathologic and genetic characteristics in cases with C9ORF72 mutations to those without. We confirmed the apparent specificity of p62 positive, TDP‐43 negative inclusions to cases with C9ORF72 mutations. In hippocampus, these inclusions correlated with hippocampal atrophy. No additional correlations were uncovered. However, this is the first report to show that although most cases with C9ORF72 mutations were TDP type B, some of the pathologic characteristics in these cases were more similar to TDP types A and C than to type B cases. These include greater cortical and hippocampal atrophy, greater ventricular dilatation, more neuronal loss and gliosis in temporal lobe and striatum, and TDP‐43 positive fine neuritic profiles in the hippocampus, implying that the C9ORF72 mutation modifies the pathologic phenotype of FTLD‐TDP type B.     

Clinical and neuropathologic heterogeneity of c9FTD/ALS associated with hexanucleotide repeat expansion in C9ORF72

Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS) are part of a disease spectrum associated with TDP-43 pathology. Strong evidence supporting this is the existence of kindreds with family members affected by FTD, ALS or mixed features of FTD and ALS, referred to as FTD-MND. Some of these families have linkage to chromosome 9, with hexanucleotide expansion mutation in a noncoding region of C9ORF72. Discovery of the mutation defines c9FTD/ALS. Prior to discovery of mutations in C9ORF72, it was assumed that TDP-43 pathology in c9FTD/ALS was uniform. In this study, we examined the neuropathology and clinical features of 20 cases of c9FTD/ALS from a brain bank for neurodegenerative disorders. Included are six patients clinically diagnosed with ALS, eight FTD, one FTD-MND and four Alzheimer-type dementia. Clinical information was unavailable for one patient. Pathologically, the cases all had TDP-43 pathology, but there were three major pathologic groups: ALS, FTLD-MND and FTLD-TDP. The ALS cases were morphologically similar to typical sporadic ALS with almost no extramotor TDP-43 pathology; all had oligodendroglial cytoplasmic inclusions. The FTLD-MND showed predominantly Mackenzie Type 3 TDP-43 pathology, and all had ALS-like pathology in motor neurons, but more extensive extramotor pathology, with oligodendroglial cytoplasmic inclusions and infrequent hippocampal sclerosis. The FTLD-TDP cases had several features similar to FTLD-TDP due to mutations in the gene for progranulin, including Mackenzie Type 1 TDP-43 pathology with neuronal intranuclear inclusions and hippocampal sclerosis. FTLD-TDP patients were older and some were thought to have Alzheimer-type dementia. In addition to the FTD and ALS clinical presentations, the present study shows that c9FTD/ALS can have other presentations, possibly related to age of onset and the presence of hippocampal sclerosis. Moreover, there is pathologic heterogeneity not only between ALS and FTLD, but also within the FTLD group. Further studies are needed to address the molecular mechanism of clinical and pathological heterogeneity of c9FTD/ALS due to mutations in C9ORF72.     

Characterization of a Family With c9FTD/ALS Associated With the GGGGCC Repeat Expansion in C9ORF72

BACKGROUND The hexanucleotide repeat in the chromosome 9 open reading frame 72 (C9ORF72) gene was recently discovered as the underlying genetic cause of many families with frontotemporal dementia (FTD) and/or amyotrophic lateral sclerosis (ALS) linked to chromosome 9 (c9FTD/ALS). We report the clinical, neuropsychologic, and neuroimaging findings of a family with the C9ORF72 mutation and clinical diagnoses bridging the FTD, parkinsonism, and ALS spectrum. OBJECTIVE To characterize the antemortem characteristics of a family with c9FTD/ALS associated with the GGGGCC repeat expansion in C9ORF72. DESIGN Clinical series. SETTING Tertiary care academic medical center. PATIENTS The members of a family affected by the mutation with features of FTD and/or ALS. MAIN OUTCOME MEASURES Clinical, neuropsychologic, and neuroimaging assessments. RESULTS All 3 examined subjects had the hexanucleotide expansion detected in C9ORF72. All had personality/behavioral changes early in the course of the disease. One case had levodopa-unresponsive parkinsonism, and 1 had ALS. Magnetic resonance imaging showed symmetric bilateral frontal, temporal, insular, and cingulate atrophy. CONCLUSIONS This report highlights the clinical and neuroimaging characteristics of a family with c9FTD/ALS. Further studies are needed to better understand the phenotypical variability and the cliniconeuroimaging-neuropathologic correlations.     

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

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

Phosphorylated and cleaved TDP‐43 in ALS,FTLD and other neurodegenerative disorders and in cellular models of TDP‐43 proteinopathy

Transactivation response (TAR) DNA‐binding protein of Mr 43 kDa (TDP‐43) is a major component of the tau‐negative and ubiquitin‐positive inclusions that characterize amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration which is now referred to as FTLD‐TDP. Concurrent TDP‐43 pathology has been reported in a variety of other neurodegenerative disorders such as Alzheimer's disease, forming a group of TDP‐43 proteinopathy. Accumulated TDP‐43 is characterized by phosphorylation and fragmentation. There is a close relationship between the pathological subtypes of FTLD‐TDP and the immunoblot pattern of the C‐terminal fragments of phosphorylated TDP‐43. These results suggest that proteolytic processing of accumulated TDP‐43 may play an important role for the pathological process. In cultured cells, transfected C‐terminal fragments of TDP‐43 are more prone to form aggregates than full‐length TDP‐43. Transfecting the C‐terminal fragment of TDP‐43 harboring pathogenic mutations of TDP‐43 gene identified in familial and sporadic ALS cases into cells enhanced the aggregate formation. Furthermore, we found that methylene blue and dimebon inhibit aggregation of TDP‐43 in these cellular models. Understanding the mechanism of phosphorylation and truncation of TDP‐43 and aggregate formation may be crucial for clarifying the pathogenesis of TDP‐43 proteinopathy and for developing useful therapeutics.     

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.     

Accumulation of phosphorylated TDP‐43 in the CNS of a patient with Cockayne syndrome

Here, we report a case of Cockayne syndrome (CS) in a Japanese man who displayed a unique pathology of phosphorylated trans‐activation response (TAR) DNA‐binding protein 43 (pTDP‐43) with abundant Rosenthal fibers. Many round pTDP‐43‐positive structures were detected throughout the CNS; however, most of them were located in two regions that also exhibited neuronal depletion: the cerebellar cortex and the inferior olivary nucleus. To a lesser extent, these aggregates were also present in the cerebellar white matter, around the subependymal regions in the brain stem, and in the spinal cord. Intraneuronal pTDP‐43 inclusions were only observed in a small number of neurons in the inferior olivary nucleus. Double‐label immunofluorescence revealed that many of the aggregates were localized to astrocytes. The observed distribution and the morphology of the pTDP‐43‐positive structures were unique and have not yet been reported. Therefore, a pTDP‐43‐related pathology may be implicated in CS as well as in other neurodegenerative diseases such as frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Whether the pathology of these diseases reflects a primary neurodegenerative process or a secondary reaction is not known.     

Pattern of ubiquilin pathology in ALS and FTLD indicates presence of C9ORF72 hexanucleotide expansion

C9ORF72-hexanucleotide repeat expansions and ubiquilin-2 (UBQLN2) mutations are recently identified genetic markers in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We investigate the relationship between C9ORF72 expansions and the clinical phenotype and neuropathology of ALS and FTLD. Genetic analysis and immunohistochemistry (IHC) were performed on autopsy-confirmed ALS (N = 75), FTLD-TDP (N = 30), AD (N = 14), and controls (N = 11). IHC for neurodegenerative disease pathology consisted of C9ORF72, UBQLN, p62, and TDP-43. A C9ORF72 expansion was identified in 19.4 % of ALS and 31 % of FTLD-TDP cases. ALS cases with C9ORF72 expansions frequently showed a bulbar onset of disease (57 %) and more rapid disease progression to death compared to non-expansion cases. Staining with C9ORF72 antibodies did not yield specific pathology. UBQLN pathology showed a highly distinct pattern in ALS and FTLD-TDP cases with the C9ORF72 expansion, with UBQLN-positive cytoplasmic inclusions in the cerebellar granular layer and extensive UBQLN-positive aggregates and dystrophic neurites in the hippocampal molecular layer and CA regions. These UBQLN pathologies were sufficiently unique to allow correct prediction of cases that were later confirmed to have C9ORF72 expansions by genetic analysis. UBQLN pathology partially co-localized with p62, and to a minor extent with TDP-43 positive dystrophic neurites and spinal cord skein-like inclusions. Our data indicate a pathophysiological link between C9ORF72 expansions and UBQLN proteins in ALS and FTLD-TDP that is associated with a highly characteristic pattern of UBQLN pathology. Our study indicates that this pathology is associated with alterations in clinical phenotype, and suggests that the presence of C9ORF72 repeat expansions may indicate a worse prognosis in ALS.     

Brain 18F-FDG and 11C-PiB PET findings in two siblings with FTD/ALS associated with the C9ORF72 repeat expansion

The C9ORF72 hexanucleotide expansion is a major pathological expansion pattern found in patients with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (C9FTD/ALS). We describe a patient in whom early clinical evaluation, MRI and fluorodeoxyglucose (FDG) positron emission tomography (PET) findings failed to definitively differentiate between FTD and Alzheimer’s disease (AD), whereas ¹¹C-Pittsburgh compound B (PiB) PET was negative for amyloid pathology. He later developed ALS symptoms, and post mortem neuropathological findings were diagnostic of FTD-ALS, while no findings suggested AD. His sister was diagnosed with FTD, and the C9ORF72 expansion was detected in both siblings. We conclude that ¹¹C-PiB PET imaging may help the early differential diagnosis between AD and FTD, including C9FTD/ALS.