An intronic G4C2 hexanucleotide repeat expansion in C9ORF72 is a major cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Several mechanisms including RNA toxicity, repeat-associated non-AUG translation mediated dipeptide protein aggregates, and haploinsufficiency of C9orf72 have been implicated in the molecular pathogenesis of this disorder. The aims of this study were to compare the use of two different Southern blot probes for detection of repeat expansions in an amyotrophic lateral sclerosis and frontotemporal lobar degeneration pathological cohort and to determine the levels of C9orf72 transcript variants and protein isoforms in patients versus control subjects. Our Southern blot studies identified smaller repeat expansions (250–1800 bp) that were only detectable with the flanking probe highlighting the potential for divergent results using different Southern blotting protocols that could complicate genotype–phenotype correlation studies. Further, we characterize a new C9orf72 antibody and show for the first time decreased C9orf72 protein levels in the frontal cortex from patients with a pathological hexanucleotide repeat expansion. These data suggest that a reduction in C9orf72 protein may be a consequence of the disease.
Amyotrophic lateral sclerosis; C9orf72; Frontotemporal dementia; Frontotemporal lobar degeneration; Repeat expansion; Southern blotting
Parkinson's disease (PD) is a neurodegenerative disorder classically characterized by the death of dopamine (DA) neurons in the substantia nigra pars compacta and by intracellular Lewy bodies composed largely of α-synuclein. Approximately 5–10% of PD patients have a familial form of Parkinsonism, including mutations in α-synuclein. To better understand the cell-type specific role of α-synuclein on DA neurotransmission, and the effects of the disease-associated A30P mutation, we generated and studied a novel transgenic model of PD. We expressed the A30P mutant form of human α-synuclein in a spatially-relevant manner from the 111 kb SNCA genomic DNA locus on a bacterial artificial chromosome (BAC) insert on a mouse null (Snca −/−) background. The BAC transgenic mice expressed α-synuclein in tyrosine hydroxylase-positive neurons and expression of either A30P α-synuclein or wildtype α-synuclein restored the sensitivity of DA neurons to MPTP in resistant Snca −/− animals. A30P α-synuclein mice showed no Lewy body-like aggregation, and did not lose catecholamine neurons in substantia nigra or locus coeruleus. However, using cyclic voltammetry at carbon-fiber microelectrodes we identified a deficit in evoked DA release in the caudate putamen, but not in the nucleus accumbens, of SNCA-A30P Snca −/− mice but no changes to release of another catecholamine, norepinephrine (NE), in the NE-rich ventral bed nucleus of stria terminalis. SNCA-A30P Snca −/− mice had no overt behavioral impairments but exhibited a mild increase in wheel-running. In summary, this refined PD mouse model shows that A30P α-synuclein preferentially perturbs the dopaminergic system in the dorsal striatum, reflecting the region-specific change seen in PD.
•SNCA A30P BAC transgenic mice recapitulate endogenous α-synuclein expression pattern•SNCA A30P BAC transgenic mice demonstrate a region specific deficit in evoked DA, but not NE, release.•Expression of A30P or WT α-syn restored the sensitivity of DA neurons to MPTP.•A30P BAC mice had no Lewy body-like aggregation or neuronal loss in SNpc or LC.•Early changes in DA neurotransmission in the absence of aggregation
Parkinson's disease; α-Synuclein; Dopamine; Norepinephrine; Voltammetry; Behavior
A hexanucleotide repeat expansion in the C9ORF72 gene has recently been shown to cause a large proportion of amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD).
We screened 4,448 patients diagnosed with ALS and 1,425 patients diagnosed with FTD drawn from diverse populations for the hexanucleotide expansion using a repeat-primed PCR assay. ALS and FTD were diagnosed according to the El Escorial and Lund-Manchester criteria respectively. Familial status was based on self-reported family history of similar neurodegenerative diseases at the time of sample collection. Haplotype data of 262 patients carrying the expansion were compared with the known Finnish founder risk haplotype across the chromosomal locus. Age-related penetrance was calculated by the Kaplan-Meier method using data from 603 individuals carrying the expansion.
The mutation was observed among 7·0% (n = 236 of 3,377) of Caucasians, 4·1% (n = 2 of 49) of African-Americans, and 8·3% (n = 6 of 72) of Hispanic individuals diagnosed with sporadic ALS, whereas the rate was 6·0% (n = 59 of 981) among Caucasians diagnosed with sporadic FTD. Among Asians, 5·0% (n = 1 of 20) of familial ALS and 66·6% (n = 2 of 3) of familial FTD cases carried the repeat expansion. In contrast, mutations were not observed among patients of Native American (n = 3 sporadic ALS), Indian (n = 31 sporadic ALS, n = 31 sporadic FTD), and Pacific Islander (n = 90 sporadic ALS) ethnicity. All patients with the repeat expansion carried, either partially or fully, the founder haplotype suggesting that the expansion occurred on a single occasion in the past (~1,500 years ago). The pathogenic expansion was non-penetrant below 35 years of age, increasing to 50·0% penetrance by 58 years of age, and was almost fully penetrant by 80 years of age.
We confirm that a common single Mendelian genetic lesion is implicated in a large proportion of sporadic and familial ALS and FTD. Testing for this pathogenic expansion will be important in the management and genetic counseling of patients with these fatal neurodegenerative diseases.
Multiple endocrine neoplasia type 1 (MEN1) is characterized by the combined occurrence of pituitary, pancreatic and parathyroid tumors showing loss of heterozygosity in the putative tumor suppressor gene MEN1. This gene encodes the protein menin, the overexpression of which inhibits cell proliferation in vitro. In this study, we conducted a preclinical evaluation of MEN1 gene therapy in pituitary tumors of Men1+/− mice, using a recombinant non-replicating adenoviral serotype 5 vector that contained the murine Men1 cDNA under control of a cytomegalovirus promoter (Men1.rAd5). Pituitary tumours in 55 Men1+/− female mice received a transauricular, intratumoral injection of Men1.rAd5 or control treatments, followed by 5-bromo-2-deoxyuridine (BrdU) in drinking water for four weeks before magnetic resonance imaging (MRI) and immunohistochemical analysis. Immediate procedure-related and four-week mortalities were similar in all groups, indicating that the adenoviral gene therapy was not associated with a higher mortality. Menin expression was higher in the Men1.rAd5-treated mice when compared to other groups. Daily proliferation rates assessed by BrdU incorporation were reduced significantly in Men1.rAd5-injected tumors relative to control treated tumors. In contrast, apoptotic rates, immune T cell response and tumor volumes remained similar in all groups. Our findings establish that MEN1 gene replacement therapy can generate menin expression in pituitary tumors, and significantly reduce tumor cell proliferation.
Neuroendocrine tumours (NETs); adenoviral vector; tumour suppressor; apoptosis; immune reaction; Men1
Accumulation of the DNA/RNA binding protein fused in sarcoma as cytoplasmic inclusions in neurons and glial cells is the pathological hallmark of all patients with amyotrophic lateral sclerosis with mutations in FUS as well as in several subtypes of frontotemporal lobar degeneration, which are not associated with FUS mutations. The mechanisms leading to inclusion formation and fused in sarcoma-associated neurodegeneration are only poorly understood. Because fused in sarcoma belongs to a family of proteins known as FET, which also includes Ewing’s sarcoma and TATA-binding protein-associated factor 15, we investigated the potential involvement of these other FET protein family members in the pathogenesis of fused in sarcoma proteinopathies. Immunohistochemical analysis of FET proteins revealed a striking difference among the various conditions, with pathology in amyotrophic lateral sclerosis with FUS mutations being labelled exclusively for fused in sarcoma, whereas fused in sarcoma-positive inclusions in subtypes of frontotemporal lobar degeneration also consistently immunostained for TATA-binding protein-associated factor 15 and variably for Ewing’s sarcoma. Immunoblot analysis of proteins extracted from post-mortem tissue of frontotemporal lobar degeneration with fused in sarcoma pathology demonstrated a relative shift of all FET proteins towards insoluble protein fractions, while genetic analysis of the TATA-binding protein-associated factor 15 and Ewing’s sarcoma gene did not identify any pathogenic variants. Cell culture experiments replicated the findings of amyotrophic lateral sclerosis with FUS mutations by confirming the absence of TATA-binding protein-associated factor 15 and Ewing’s sarcoma alterations upon expression of mutant fused in sarcoma. In contrast, all endogenous FET proteins were recruited into cytoplasmic stress granules upon general inhibition of Transportin-mediated nuclear import, mimicking the findings in frontotemporal lobar degeneration with fused in sarcoma pathology. These results allow a separation of fused in sarcoma proteinopathies caused by FUS mutations from those without a known genetic cause based on neuropathological features. More importantly, our data imply different pathological processes underlying inclusion formation and cell death between both conditions; the pathogenesis in amyotrophic lateral sclerosis with FUS mutations appears to be more restricted to dysfunction of fused in sarcoma, while a more global and complex dysregulation of all FET proteins is involved in the subtypes of frontotemporal lobar degeneration with fused in sarcoma pathology.
FUS; TAF15; EWS; amyotrophic lateral sclerosis; frontotemporal dementia
Mutations in the gene encoding the fused in sarcoma (FUS) protein are responsible for ~3% of familial amyotrophic lateral sclerosis (ALS) and <1% of sporadic ALS (ALS-FUS). Descriptions of the associated neuropathology are few and largely restricted to individual case reports. To better define the neuropathology associated with FUS mutations, we have undertaken a detailed comparative analysis of six cases of ALS-FUS that include sporadic and familial cases, with both juvenile and adult onset, and with four different FUS mutations. We found significant pathological heterogeneity among our cases, with two distinct patterns that correlated with the disease severity and the specific mutation. Frequent basophilic inclusions and round FUS-immunoreactive (FUS-ir) neuronal cytoplasmic inclusions (NCI) were a consistent feature of our early-onset cases, including two with the p.P525L mutation. In contrast, our late-onset cases, that included two with the p.R521C mutation, had tangle-like NCI and numerous FUS-ir glial cytoplasmic inclusions. Double-labeling experiments demonstrated that many of the glial inclusions were in oligodendrocytes. Comparison with the neuropathology of cases of frontotemporal lobar degeneration with FUS-ir pathology showed significant differences and suggests that FUS mutations are associated with a distinct pathobiology.
fused in sarcoma; FUS; amyotrophic lateral sclerosis; ALS; basophilic inclusions
We aimed to accurately estimate the frequency of a hexanucleotide repeat expansion in C9orf72 that has been associated with a large proportion of cases of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD).
We screened 4448 patients diagnosed with ALS (El Escorial criteria) and 1425 patients with FTD (Lund-Manchester criteria) from 17 regions worldwide for the GGGGCC hexanucleotide expansion using a repeat-primed PCR assay. We assessed familial disease status on the basis of self-reported family history of similar neurodegenerative diseases at the time of sample collection. We compared haplotype data for 262 patients carrying the expansion with the known Finnish founder risk haplotype across the chromosomal locus. We calculated age-related penetrance using the Kaplan-Meier method with data for 603 individuals with the expansion.
In patients with sporadic ALS, we identified the repeat expansion in 236 (7·0%) of 3377 white individuals from the USA, Europe, and Australia, two (4·1%) of 49 black individuals from the USA, and six (8·3%) of 72 Hispanic individuals from the USA. The mutation was present in 217 (39·3%) of 552 white individuals with familial ALS from Europe and the USA. 59 (6·0%) of 981 white Europeans with sporadic FTD had the mutation, as did 99 (24·8%) of 400 white Europeans with familial FTD. Data for other ethnic groups were sparse, but we identified one Asian patient with familial ALS (from 20 assessed) and two with familial FTD (from three assessed) who carried the mutation. The mutation was not carried by the three Native Americans or 360 patients from Asia or the Pacific Islands with sporadic ALS who were tested, or by 41 Asian patients with sporadic FTD. All patients with the repeat expansion had (partly or fully) the founder haplotype, suggesting a one-off expansion occurring about 1500 years ago. The pathogenic expansion was non-penetrant in individuals younger than 35 years, 50% penetrant by 58 years, and almost fully penetrant by 80 years.
A common Mendelian genetic lesion in C9orf72 is implicated in many cases of sporadic and familial ALS and FTD. Testing for this pathogenic expansion should be considered in the management and genetic counselling of patients with these fatal neurodegenerative diseases.
Full funding sources listed at end of paper (see Acknowledgments).
The concurrence of multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS) is exceedingly rare and the pathological features have not been examined extensively. Here we describe the key pathological features of a 40 year old man with pathologically confirmed concurrent MS and ALS.
This is the most pathologically illustrative case of coincident MS and ALS demonstrating inflammatory and neurodegenerative features characteristic of each disease, and is the first to exhibit the presence of TDP-43 inclusions in this clinical entity. The intricate relationship between neuroinflammation and neurodegeneration in these diseases is discussed.
multiple sclerosis; amyotrophic lateral sclerosis; neuropathology; inflammation; neurodegeneration
Reduced expression of the survival motor neuron (SMN) gene causes the childhood motor neuron disease spinal muscular atrophy (SMA). Low levels of ubiquitously expressed SMN protein result in the degeneration of lower motor neurons, but it remains unclear whether other regions of the nervous system are also affected. Here we show that reduced levels of SMN lead to impaired perinatal brain development in a mouse model of severe SMA. Regionally selective changes in brain morphology were apparent in areas normally associated with higher SMN levels in the healthy postnatal brain, including the hippocampus, and were associated with decreased cell density, reduced cell proliferation and impaired hippocampal neurogenesis. A comparative proteomics analysis of the hippocampus from SMA and wild-type littermate mice revealed widespread modifications in expression levels of proteins regulating cellular proliferation, migration and development when SMN levels were reduced. This study reveals novel roles for SMN protein in brain development and maintenance and provides the first insights into cellular and molecular pathways disrupted in the brain in a severe form of SMA.
Leucine rich repeat kinase 2 (LRRK2) mutations are the most common genetic cause of Parkinson's disease (PD) although LRRK2 function remains unclear. We report a new role for LRRK2 in regulating autophagy and describe the recruitment of LRRK2 to the endosomal–autophagic pathway and specific membrane subdomains. Using a novel human genomic reporter cellular model, we found LRRK2 to locate to membrane microdomains such as the neck of caveolae, microvilli/filopodia and intraluminal vesicles of multivesicular bodies (MVBs). In human brain and in cultured human cells LRRK2 was present in cytoplasmic puncta corresponding to MVBs and autophagic vacuoles (AVs). Expression of the common R1441C mutation from a genomic DNA construct caused impaired autophagic balance evident by the accumulation of MVBs and large AVs containing incompletely degraded material and increased levels of p62. Furthermore, the R1441C mutation induced the formation of skein-like abnormal MVBs. Conversely, LRRK2 siRNA knockdown increased autophagic activity and prevented cell death caused by inhibition of autophagy in starvation conditions. The work necessitated developing a new, more efficient recombineering strategy, which we termed Sequential insertion of Target with ovErlapping Primers (STEP) to seamlessly fuse the green fluorescent protein-derivative YPet to the human LRRK2 protein in the LRRK2 genomic locus carried by a bacterial artificial chromosome. Taken together our data demonstrate the functional involvement of LRRK2 in the endosomal–autophagic pathway and the recruitment to specific membrane microdomains in a physiological human gene expression model suggesting a novel function for this important PD-related protein.
Classical Parkinson's disease (PD) is characterized by the appearance of Lewy bodies (LBs) in affected brain regions, showing mostly compact alpha-synuclein deposition, in contrast with punctate or granular deposition, hypothesized to represent early stages of aggregation. Leucine-rich repeat kinase 2 (LRRK2) is the commonest mutated gene in inherited and idiopathic PD. LRRK2 mutation carriers display a diverse neuropathology, including alpha-synuclein and tau inclusions, suggesting an upstream role for LRRK2 in protein aggregation. We studied LRRK2 expression throughout the normal human brain with three different antibodies. We also examined the pattern of LRRK2 expression in relation to alpha-synuclein aggregation and LB formation in the brainstem of sporadic LB disease. Physiological LRRK2 expression was not restricted to regions preferentially affected in PD and LRRK2 often localised to the nuclear envelope in addition to the known cytoplasmic expression. In PD, we were able to consistently detect LRRK2 in the halo of a minority (~10%) of nigral LBs using three different antibodies. Only one antibody detected LRRK2 in the core of ~80% of classic LBs. In the lower brainstem, most notably in the dorsal motor nucleus of the vagus, we found previously unrecognised LRRK2 labelling of complex globular lesions, filled with LB-like-matter showing a punctate or granular staining for alpha-synuclein. This was often accompanied by strong LRRK2 expression within dystrophic neurites. Our findings confirm widespread physiological LRRK2 expression in the human brain and suggest an association of LRRK2 with possible early-stage alpha-synuclein pathology in the brainstem of PD.
LRRK2; dardarin; alpha-synuclein; Parkinson's disease; brainstem; Lewy body
Mitochondrial DNA mutations are an important cause of neurological disease. The clinical presentation is very varied in terms of age of onset and different neurological signs and symptoms. The clinical course varies considerably but in many patients there is a progressive decline, and in some evidence of marked neurodegeneration. Our understanding of the mechanisms involved is limited due in part to limited availability of animal models of disease. However, studies on human post-mortem brains, combined with clinical and radiological studies, are giving important insights into specific neuronal involvement.
Spinal muscular atrophy is a severe motor neuron disease caused by inactivating mutations in the SMN1 gene leading to reduced levels of full-length functional SMN protein. SMN is a critical mediator of spliceosomal protein assembly, and complete loss or drastic reduction in protein leads to loss of cell viability. However, the reason for selective motor neuron degeneration when SMN is reduced to levels which are tolerated by all other cell types is not currently understood. Widespread splicing abnormalities have recently been reported at end-stage in a mouse model of SMA, leading to the proposition that disruption of efficient splicing is the primary mechanism of motor neuron death. However, it remains unclear whether splicing abnormalities are present during early stages of the disease, which would be a requirement for a direct role in disease pathogenesis. We performed exon-array analysis of RNA from SMN deficient mouse spinal cord at 3 time points, pre-symptomatic (P1), early symptomatic (P7), and late-symptomatic (P13). Compared to littermate control mice, SMA mice showed a time-dependent increase in the number of exons showing differential expression, with minimal differences between genotypes at P1 and P7, but substantial variation in late-symptomatic (P13) mice. Gene ontology analysis revealed differences in pathways associated with neuronal development as well as cellular injury. Validation of selected targets by RT–PCR confirmed the array findings and was in keeping with a shift between physiologically occurring mRNA isoforms. We conclude that the majority of splicing changes occur late in SMA and may represent a secondary effect of cell injury, though we cannot rule out significant early changes in a small number of transcripts crucial to motor neuron survival.
The identification of mutations in the Survival Motor Neuron (SMN) gene as the cause of the severe motor neuron disorder spinal muscular atrophy is one of a number of discoveries implicating selective motor neuron vulnerability to defects in processing of RNA and its associated ribonucleoprotein complexes. An unresolved issue is whether loss of the general cellular function of SMN in spliceosomal assembly, which is predicted to result in widespread defects in mRNA splicing, is directly responsible for motor neuron death. We have used exon-specific microarrays to assess the degree of altered splicing in the spinal cord in a mouse model of SMA. Our finding that the vast majority of splicing changes are a late feature of the disease and may represent a shift to alternative isoform expression, rather than loss of splicing fidelity, provides evidence that widespread splicing disturbance is not a primary feature of the disease pathogenesis but a secondary effect of cell injury in a late phase of the disease. However, our study cannot rule out a role for subtle early changes in one or a few transcripts crucial to motor neuron survival expressed at low levels or in only in a sub-population of spinal cord cells.
Brain-specific homing and direct interactions with the neural substance are prominent hypotheses for brain metastasis formation and a modern manifestation of Paget's “seed and soil” concept. However, there is little direct evidence for this “neurotropic” growth in vivo. In contrast, many experimental studies have anecdotally noted the propensity of metastatic cells to grow along the exterior of pre-existing vessels of the CNS, a process termed vascular cooption. These observations suggest the “soil” for malignant cells in the CNS may well be vascular, rather than neuronal. We used in vivo experimental models of brain metastasis and analysis of human clinical specimens to test this hypothesis. Indeed, over 95% of early micrometastases examined demonstrated vascular cooption with little evidence for isolated neurotropic growth. This vessel interaction was adhesive in nature implicating the vascular basement membrane (VBM) as the active substrate for tumor cell growth in the brain. Accordingly, VBM promoted adhesion and invasion of malignant cells and was sufficient for tumor growth prior to any evidence of angiogenesis. Blockade or loss of the β1 integrin subunit in tumor cells prevented adhesion to VBM and attenuated metastasis establishment and growth in vivo. Our data establishes a new understanding of CNS metastasis formation and identifies the neurovasculature as the critical partner for such growth. Further, we have elucidated the mechanism of vascular cooption for the first time. These findings may help inform the design of effective molecular therapies for patients with fatal CNS malignancies.
Redistribution of nuclear TAR DNA binding protein 43 (TDP-43) to the cytoplasm and ubiquitinated inclusions of spinal motor neurons and glial cells is characteristic of amyotrophic lateral sclerosis (ALS) pathology. Recent evidence suggests that TDP-43 pathology is common to sporadic ALS and familial ALS without SOD1 mutation, but not SOD1-related fALS cases. Furthermore, it remains unclear whether TDP-43 abnormalities occur in non-ALS forms of motor neuron disease. Here, we characterise TDP-43 localisation, expression levels and post-translational modifications in mouse models of ALS and spinal muscular atrophy (SMA).
TDP-43 mislocalisation to ubiquitinated inclusions or cytoplasm was notably lacking in anterior horn cells from transgenic mutant SOD1G93A mice. In addition, abnormally phosphorylated or truncated TDP-43 species were not detected in fractionated ALS mouse spinal cord or brain. Despite partial colocalisation of TDP-43 with SMN, depletion of SMN- and coilin-positive Cajal bodies in motor neurons of affected SMA mice did not alter nuclear TDP-43 distribution, expression or biochemistry in spinal cords.
These results emphasise that TDP-43 pathology characteristic of human sporadic ALS is not a core component of the neurodegenerative mechanisms caused by SOD1 mutation or SMN deficiency in mouse models of ALS and SMA, respectively.