Natasha Luquin

TDP‐43 neuropathology is similar in sporadic amyotrophic lateral sclerosis with or without TDP‐43 mutations

Patients with both sporadic amyotrophic lateral sclerosis (SALS) and familial ALS (FALS) without SOD1 mutations have TDP-43 inclusions in their motor neurones and glia [1,2]. Of further interest to the pathogenesis of ALS has been the finding of mutations in the TDP-43 gene in patients with either FALS or SALS [3–7]. However, only 7 of 1206 SALS patients (0.6%) studied so far have been found to have TDP-43 mutations, with some series reporting no mutations [8,9]. In the majority of SALS patients who do not have TDP-43 mutations the relevance of TDP-43 inclusions remains unclear. Neuropathological examination of four FALS patients with TDP-43 mutations has indicated that the distribution of TDP-43 pathology was more widespread than in most nonmutant TDP-43 ALS patients [5,7]. We describe for the first time the neuropathology in a patient with SALS who had a TDP-43 mutation. A predominant loss of lower motor neurones was found, and the pattern of TDP-43 pathology was similar to that of SALS patients without TDP-43 mutations. The patient was a white civil engineer of Scottish and Irish descent, born in New Zealand, who emigrated to Australia aged 60 years. When aged 64 years he noticed weight loss and wasting of his muscles. He had a long history of leg cramps, which become more severe 6 months before presentation. When aged 65 years he was diagnosed with thyrotoxicosis, which was treated with radioactive iodine and propylthiouracil. His father and mother both died aged 89 years, his mother (an only child) having survived colon and breast cancer. He had two paternal uncles, both of whom died young, one in his teens and the other in his 30s, but the causes of death were not known. He had one brother aged 64 years. No family member suffered from any neurological disease. On examination, he had widespread fasciculations and wasting and weakness of muscles in the upper and lower limbs. Upperand lower-limb tendon reflexes were exaggerated, but no increase in tone was noted and the plantar responses were flexor. MRI of his brain and cervical spinal cord showed no abnormality. Upperand lower-limb sensory and motor studies had normal amplitudes and conduction velocities with no evidence of conduction block or dispersion. There was mild prolongation of F wave latencies. On needle EMG there were multiform fasciculations in multiple muscles in all four limbs with evidence of chronic partial denervation in most muscles. He donated a blood sample to the Australian Motor Neuron Disease DNA Bank and predonated his brain and spinal cord to the Motor Neuron Disease Research Tissue Bank of Victoria. Cognition, affect and bulbar function remained intact. He became progressively weaker, had respiratory difficulties and died aged 70 years. At post-mortem (6-h delay) half of the cerebrum, cerebellum and brain stem, and segments of cervical, thoracic, lumbar and sacral spinal cord, were fixed in 10% formalin. Coronal sections of the hemi-cerebrum, parasagittal sections of the hemi-cerebellum and transverse sections of the hemi-brain stem showed no macroscopic abnormalities. The spinal anterior roots were atrophic. No macroscopic abnormalities were seen on transverse sections of the spinal cord. Standard blocks of formalin-fixed tissue were paraffin-embedded, sectioned at 6 mm and stained with haematoxylin and eosin, Nissl and Luxol fast blue. Immunostaining was with rabbit antiTARDBP (10782-1-AP, Proteintech Group, Chicago, IL, USA), rabbit anti-ubiquitin (Z0458, DakoCytomation, Carpinteria, CA, USA) and mouse anti-human CD68 Clone KP1 (M0814, DakoCytomation), all using the avidin-biotin technique and haematoxylin counterstaining. The remaining parts of the brain and spinal cord that had not been fixed in formalin were kept frozen at -70°C. The project was approved by the Human Ethics Committee of the Sydney South West Area Health Service. No degeneration of the lateral or anterior corticospinal tracts was seen on myelin staining (Figure 1A). A slight increase in macrophage number was seen in the corticospinal tracts in the caudal spinal cord on haematoxylin and eosin sections, and this was confirmed on CD68 immunostaining (Figure 1B,C) [10]. Loss of anterior horn motor neurones was severe at all levels of the spinal cord. About 10% of remaining spinal motor neurones contained either filamentous or round TDP-43 cytoplasmic

Genetic variants in the promoter of TARDBP in sporadic amyotrophic lateral sclerosis

All patients with sporadic amyotrophic lateral sclerosis (SALS) have TDP-43 inclusions in their motor neurons, suggesting this protein plays a major role in the disease. Coding mutations in the gene for TDP-43, TARDBP, have been found in only a few patients with SALS. However, the non-coding regulatory regions of TARDBP have not yet been examined in SALS. We therefore sequenced both coding and non-coding regions of TARDBP in 46 tissue-banked SALS brains (brain DNA was used to detect somatic mutations). Non-coding variants (in the promoter or intron 1) were detected in 16 patients (35%) and coding variants in 4 (9%). Two known promoter variants were found more frequently in SALS patients than in controls. Two other variants, found in one patient each but not in controls, have potential regulatory functions. In addition, a novel exon 2 change with predicted functional effects was found in one patient. In summary, variants in the promoter and other non-coding regions of TARDBP may disturb the regulation of this gene in some patients with SALS.

An analysis of the entire SOD1 gene in sporadic ALS

Mutations in the superoxide dismutase 1 gene (SOD1) are associated with familial ALS but the role of SOD1 in sporadic ALS (SALS) is unclear. We therefore sequenced the entire SOD1 gene in 23 patients with SALS. DNA was extracted from frozen pre-frontal cerebral cortex and from blood. The 5 exons, 4 introns and 1 kb upstream and downstream of SOD1 were sequenced. Novel genetic variants were found in 30% (7 of 23) brains and known variants in 91% (21 of 23) brains from patients with SALS. Two novel variants found in SALS patients and not controls were located in the SOD1 promoter and intron 1, with the promoter variant having potential functional implications. A previously described silent variant in exon 5 in one SALS patient appears to abolish an exonic splicing enhancer. All changes found in brain DNA were also found in blood DNA. In conclusion, sequencing the entire SOD1 gene revealed 3 variants in SALS patients that were not detected in controls. Although no unequivocal mutations were found, some of these variants have potential consequences for SALS pathogenesis.

Looking for differences in copy number between blood and brain in sporadic amyotrophic lateral sclerosis

Introduction: Most analyses of blood DNA in sporadic neuromuscular disorders have been inconclusive. This may be because some genetic variants occur only in brain tissue. We therefore looked for copy number variants (CNVs) in both blood and brain in patients with sporadic amyotrophic lateral sclerosis (SALS). Methods: Genome‐wide CNVs were compared in blood and brain from 32 SALS patients and from 26 normal (control) brains, using Affymetrix 6.0 arrays. Results: There were 410 CNVs present in brain but not blood (somatic CNVs) in 94% of the patients (median 8 CNVs per patient). Twenty‐four of the somatic CNVs were rare, were not found in control brains, and overlapped with genes. Conclusions: Brain‐specific CNVs may be common and appear to be present in a proportion of patients with SALS. The more detailed copy number analysis that is becoming available with massively parallel sequencing may uncover brain‐specific CNVs that underlie some cases of SALS. Muscle Nerve, 2011

The endocannabinoid system in cardiovascular function: novel insights and clinical implications

AbstractRationaleCardiovascular disease is now recognized as the number one cause of death in the world, and the size of the population at risk continues to increase rapidly. The dysregulation of the endocannabinoid (eCB) system plays a central role in a wide variety of conditions including cardiovascular disorders. Cannabinoid receptors, their endogenous ligands, as well as enzymes conferring their synthesis and degradation, exhibit overlapping distributions in the cardiovascular system. Furthermore, the pharmacological manipulation of the eCB system has effects on blood pressure, cardiac contractility, and endothelial vasomotor control. Growing evidence from animal studies supports the significance of the eCB system in cardiovascular disorders. ObjectiveTo summarize the literature surrounding the eCB system in cardiovascular function and disease and the new compounds that may potentially extend the range of available interventions.ResultsDrugs targeting CB1R, CB2R, TRPV1 and PPARs are proven effective in animal models mimicking cardiovascular disorders such as hypertension, atherosclerosis and myocardial infarction. Despite the setback of two clinical trials that exhibited unexpected harmful side-effects, preclinical studies are accelerating the development of more selective drugs with promising results devoid of adverse effects. ConclusionOver the last years, increasing evidence from basic and clinical research supports the role of the eCB system in cardiovascular function. Whereas new discoveries are paving the way for the identification of novel drugs and therapeutic targets, the close cooperation of researchers, clinicians and pharmaceutical companies is needed to achieve successful outcomes.

A Novel TARDBP insertion/deletion mutation in the flail arm variant of amyotrophic lateral sclerosis

Abstract Phenotypic variation in amyotrophic lateral sclerosis (ALS) is common, and one atypical form is the flail arm variant (FAV). Some classic ALS patients carry TARDBP mutations, and so we sought to establish whether TARDBP mutations are also present in the FAV of ALS. Mutation analysis of TARDBP, the gene encoding TDP-43, was performed in cohorts of classic and FAV ALS patients. An analysis of mutation effects was performed in patient fibroblasts. Results showed that a novel heterozygous in-frame insertion/deletion (indel), c.1158_1159delAT; c.1158_1159insCACCAACC, was identified in a highly conserved region encoding the glycine-rich area of TDP-43 in a patient with FAV. This indel was confirmed in the probands mother, an obligate carrier, and was absent from 480 ethnically-matched control individuals. Transcription of the mutant allele was confirmed. Under induced stress, indel-mutant fibroblasts showed a loss of normal nuclear TDP-43 immunoreactivity and formation of cytoplasmic inclusions of TDP-43, consistent with features seen in affected neurons. In conclusion, TARDBP missense mutations have previously been reported in classic ALS and frontotemporal lobar degeneration. The identification of a TARDBP indel mutation in a patient with FAV extends the spectrum of mutations and further supports the role of TDP-43 in a range of neurodegenerative phenotypes.

DHPLC can be used to detect low-level mutations in amyotrophic lateral sclerosis.

Somatic mutations have been suggested as a cause of sporadic amyotrophic lateral sclerosis (SALS). These mutations can be difficult to detect since they may involve only a small percentage of cells within the tissue, so we devised a method to detect low mutation levels in brain DNA. Different proportions of a known SOD1 mutation were prepared to determine the sensitivity of DHPLC. The fraction containing the mutant signal was collected and re-amplified (‘enriched’) to increase sensitivity and to dideoxy sequence the mutation. The combined technique was used to screen all exons and the promoter of SOD1 in 23 SALS brains. DHPLC could detect a known SOD1 mutation in 5% of a sample of brain tissue. Using our enrichment technique doubled the height of the mutant sequencing signal, which allowed identification of an unknown mutation in 10% of brain tissue. No SOD1 mutations were found in the SALS brains using this technique. In conclusion, combining DHPLC and sequencing doubles the sensitivity of sequencing alone and can detect low levels of known and unknown mutations in brain DNA. No SALS SOD1 somatic mutations were detected, but DHPLC would be useful in looking for somatic mutations in other SALS candidate genes.

An approach to finding brain-situated mutations in sporadic Parkinson's disease.

Sporadic Parkinsons disease (PD) is thought to have a major genetic component, but the variants involved remain mostly unknown. One possible reason for the difficulty in finding mutations underlying PD is that rare predominantly brain-situated somatic mutations underlie the disease; these mutations would be missed by analysing blood DNA only. To test the feasibility of looking for somatic mutations in PD brain tissue, we compared copy number variants (CNVs) between 8 PD and 26 control brains using Affymetrix 6.0 arrays. The median number of CNVs per brain, and the overall proportion of amplifications and deletions, were similar in PD and control brains. In 7 of the 8 PD brains, however, a total of 45 CNVs were found that were not present in control brains. Twelve of these CNVs overlapped with one or more genes, some of which are involved in pathways suspected in the pathogenesis of PD, or are rare. This study shows that PD brain CNVs can be detected, and raises the possibility that brain-situated mutations could underlie some cases of PD. A method of undertaking a definitive study of brain somatic mutations in PD, using massively parallel sequencing and multiple tissues, is suggested.

Choroid plexitis as a unique neurological manifestation in granulomatosis with polyangiitis (Wegener's disease).

A 70-year-old man presented with a 3-day history of nausea, vomiting and constitutional symptoms (fatigue and anorexia). During hospitalization he developed diplopia. Cranial magnetic resonance imaging with gadolinium (Figure 1a) showed inflammation of the entire choroid plexus. A diagnosis of granulomatosis with polyangiitis (GPA) was made based …