Monocytes of patients with amyotrophic lateral sclerosis linked to gene mutations display altered TDP-43 subcellular distribution.

AIMS: Cytoplasmic accumulation of the nuclear protein transactive response DNA-binding protein 43 (TDP-43) is an early determinant of motor neuron degeneration in most amyotrophic lateral sclerosis (ALS) cases. We previously disclosed this accumulation in circulating lymphomonocytes (CLM) of ALS patients with mutant TARDBP, the TDP-43-coding gene, as well as of a healthy individual carrying the parental TARDBP mutation. Here, we investigate TDP-43 subcellular localization in CLM and in the constituent cells, lymphocytes and monocytes, of patients with various ALS-linked mutant genes. METHODS: TDP-43 subcellular localization was analysed with western immunoblotting and immunocytofluorescence in CLM of healthy controls (n = 10), patients with mutant TARDBP (n = 4, 1 homozygous), valosin-containing protein (VCP; n = 2), fused in sarcoma/translocated in liposarcoma (FUS; n = 2), Cu/Zn superoxide dismutase 1 (SOD1; n = 6), chromosome 9 open reading frame 72 (C9ORF72; n = 4), without mutations (n = 5) and neurologically unaffected subjects with mutant TARDBP (n = 2). RESULTS: TDP-43 cytoplasmic accumulation was found (P < 0.05 vs. controls) in CLM of patients with mutant TARDBP or VCP, but not FUS, in line with TDP-43 subcellular localization described for motor neurons of corresponding groups. Accumulation also characterized CLM of the healthy individuals with mutant TARDBP and of some patients with mutant SOD1 or C9ORF72. In 5 patients, belonging to categories described to carry TDP-43 mislocalization in motor neurons (3 C9ORF72, 1 TARDBP and 1 without mutations), TDP-43 cytoplasmic accumulation was not detected in CLM or in lymphocytes but was in monocytes. CONCLUSIONS: In ALS forms characterized by TDP-43 mislocalization in motor neurons, monocytes display this alteration, even when not manifest in CLM. Monocytes may be used to support diagnosis, as well as to identify subjects at risk, of ALS and to develop/monitor targeted treatments.

Progranulin gene variability increases the risk for primary progressive multiple sclerosis in males.

Progranulin (GRN) gene variability has been analyzed in a sample of 354 patients with multiple sclerosis (MS) compared with 343 controls. No significant differences were observed, but by stratifying according to MS subtypes, a significant increased frequency of the rs2879096 TT genotype was found in primary progressive MS (PPMS) patients versus controls (16.0 vs 3.5%, P=0.023, odds ratio (OR) 5.2, 95% confidence interval (CI) 1.2-21.4). In addition, in PPMS, an association with the C allele of rs4792938 was observed (55.3 vs 33.5%, P=0.011, OR 2.4, 95% CI 1.2-4.7). An independent population was studied as replication, failing to confirm results previously obtained. Stratifying according to gender, an association with rs4792938 C allele was found in male PPMS patients compared with controls (40.7 vs 26.9%, P=0.002, OR 1.87, 95% CI 1.2-2.8). An association with the rs2879096T allele was observed (29.2 in patients compared with 18.9% in controls, P=0.012, OR 1.77, 95% CI 1.1-2.8). Haplotype analysis showed that TC haplotype frequency is increased in PPMS male patients compared with male controls (25.7 vs 16.6%; P=0.02, OR 1.69, 95% CI 1.1-2.7), whereas the respective GC haplotype seems to exert a protective effect, as its frequency is decreased in patients compared with controls (55.8% vs 70.9%; P=0.001, OR 0.52, 95% CI 0.4-0.8). Therefore, GRN haplotypes likely influence the risk of developing PPMS in males.

Kappa free light chains index in the differential diagnosis of Multiple Sclerosis from Neuromyelitis optica spectrum disorders and other immune-mediated central nervous system disorders.

The K free light chains index (K-FLC index) has been proposed as an alternative test for intrathecal immunoglobulin synthesis in MS diagnosis. Aim of the study was to assess the accuracy of the K-FLC index in differentiating MS from other immune-mediated CNS disorders and NMOSD. Data were available from a cohort of 371 patients. K-FLC index was significantly higher in MS: MS mean K-FLC index 90.897 ± 134.198; NMOSD 17.992 ± 15.103; other immune-mediated CNS disorders 12.568 ± 24.440. The overall diagnostic accuracy of the K-FLC index was similar to intrathecal oligoclonal bands detection. However, as a quantitative variable, K-FLC index allowed easier discrimination of MS from other immune-mediated CNS disorders: highest K-FLC index values (> 100) were observed almost only in MS and are therefore strongly predictive of MS, in patients with the appropriate clinical presentation.

The NOS3 G894T (Glu298Asp) polymorphism is a risk factor for frontotemporal lobar degeneration.

BACKGROUND AND AIMS: Neuronal nitric oxide synthase (NOS)1 C276T polymorphism was shown to increase the risk for frontotemporal lobar degeneration (FTLD). In the brain, both NOS1 and NOS3 (endothelial isoform) have been detected. The distribution of NOS3 G894T (Glu298Asp) and T-786C single nucleotide polymorphisms (SNPs) was analyzed in a population of 222 patients with FTLD compared with 218 age-matched controls to determine whether they could influence the susceptibility to develop the disease. RESULTS: A statistically significant increased frequency of the NOS3 G894T SNP was observed in patients as compared with controls (40.0 vs. 31.4%, P = 0.011, OR: 1.65, CI: 1.13-2.42). Conversely, the distribution of the T-786C SNP was similar in patients and controls. No differences were observed stratifying according to gender. DISCUSSION: The NOS3 G894T polymorphism likely acts as risk factor for sporadic FTLD, but studies in larger populations are needed to confirm these preliminary findings.

DCUN1D1 is a risk factor for frontotemporal lobar degeneration.

BACKGROUND AND PURPOSE: Frontotemporal lobar degeneration (FTLD) is considered as a proteinopathy; therefore, it is conceivable that genes encoding for factors involved in protein misfolding and/or degradation could play a role in its pathogenesis. METHODS: An association study of defective in cullin neddylation 1 (DCN-1)-domain containing 1 (DCUN1D1), which is involved in protein degradation, was carried out in a population of 220 patients with FTLD as compared with 229 age-matched controls. RESULTS: A statistically significant increased frequency of the GG genotype of the DCUN1D1 rs4859146 single nucleotide polymorphism (SNP) was observed in patients compared with controls (6.9 vs. 1.7%, P = 0.011, adjusted OR: 4.39, 95% CI: 1.40-13.78). Stratifying according to the clinical syndrome, significant differences were observed between the behavioral variant of frontotemporal dementia and controls (GG frequency: 6.3 vs. 1.7%, P = 0.02, OR:4.0, 95%, CI = 1.24-12.92), as well as between patients with progressive aphasia compared with controls (15.4 vs. 1.7%, P = 0.014, OR = 11.30, 95%, CI = 1.63-78.45), but not in patients with SD versus controls (8.3 vs. 1.7%, P = 0.18, OR = 5.24, 95% C.I. = 0.45-60.63). No significant differences in allelic and genotypic frequencies of the DCUN1D1 rs4859147 SNP were found. CONCLUSIONS: The GG genotype of the DCUN1D1 rs4859147 SNP represents a risk factor for the development of FTLD, increasing the risk of about fourfold.

Inflammatory responses in Multiple Sclerosis normal-appearing white matter and in non-immune mediated neurological conditions with wallerian axonal degeneration: A comparative study.

Inflammatory-like changes in the white matter (WM) are commonly observed in conditions of axonal degeneration by different etiologies. This study is a systematic comparison of the principal features of the inflammatory-like changes in the WM in different pathological conditions characterized by axonal damage/degeneration, focusing in particular on Multiple Sclerosis (MS) normal-appearing white matter (NAWM) compared to non immune-mediated disorders. The study was performed on sections of NAWM from 15 MS cases, 11 cases of non immune-mediated disorders with wallerian axonal degeneration (stroke, trauma, amyotrophic lateral sclerosis), 3 cases of viral encephalitis, 6 control cases. Common features of the inflammatory-like changes observed in all of the conditions of WM pathology were diffuse endothelial expression of VCAM-1, microglial activation with expression of M2 markers, increased expression of sphingosine receptors. Inflammation in MS NAWM was characterized, compared to non immune-mediated conditions, by higher VCAM-1 expression, higher density of perivascular lymphocytes, focal perivascular inflammation with microglial expression of M1 markers, ongoing acute axonal damage correlating with VCAM-1 expression but not with microglia activation. Inflammatory changes in MS NAWM share all the main features observed in the WM in non immune-mediated conditions with wallerian axonal degeneration (with differences to a large extent more quantitative than qualitative), but with superimposition of disease-specific perivascular inflammation and ongoing acute axonal damage.

Morphophenotype of medulloblastoma in children and adults. The size of nuclei.

OBJECTIVE: Uniform cells with round, regular nuclei characterize the typical histologic aspect of medulloblastoma. Enlargement of nuclei distinguishes the large-cell medulloblastoma variant and is associated with a poor prognosis in pediatric medulloblastomas. The aim of the present study was to compare the size of nuclei between pediatric and adult medulloblastomas by a morphometric analysis. MATERIAL AND METHODS: In 79 neurosurgical specimens of cerebellar medulloblastomas, the maximum nuclear diameter of the largest nuclei was measured. Measurements were performed with a digital-image analysis system. The measure of the maximum diameter was chosen in order to reduce the split cell error. RESULTS: The difference between the mean values in children and adults was statistically significant (p = 0,001). The distribution of maximum values measured in each case had two distinct peaks in the two age groups, in 3.5% of adult cases and in more than 30% of pediatric cases the maximum nuclear size was superior to 12 microm. CONCLUSIONS: The present results show that nuclei of tumor cells in pediatric medulloblastomas are larger than those in adult medulloblastomas and confirm that the phenotype of medulloblastoma is different in the two age groups. Distinct genetic events can, thus, underlie medulloblastoma in childhood and adult age, the prognostic role of genetic variables can differ by age.

Ubiquitin in motor neuron disease: study at the light and electron microscope.

Several neurodegenerative diseases, including motor neuron disease (MND), are characterized by formation of abnormal cytoskeleton-derived inclusions which contain ubiquitin (Ubq). We have studied the distribution of Ubq in 26 cases of MND with light and electron microscopic immunocytochemistry. Ubiquitin-positive inclusions were found in neurons of anterior horns in most cases of amyotrophic lateral sclerosis (ALS) but were not present in other forms of MND. Ubiquitin immunoreactivity was observed in 10-15 nm intraneuronal filaments, which were not stained by antibodies to neurofilaments, and on dense bodies of dystrophic neurites throughout the neuropil of anterior horns and pyramidal tracts. Data analysis showed a trend toward lower percentage of Ubq-positive neurons in cases with longer duration of illness or lower number of neurons. A high percentage of Ubq-positive inclusions occurred in cases with an aggressive clinical course, suggesting that ubiquitination takes place at early stages of the disease.

Early symptom progression rate is related to ALS outcome: a prospective population-based study.

OBJECTIVE: To define the factors related to ALS outcome in a population-based, prospective survey. METHODS: The 221 patients (120 men and 101 women) listed in the Piemonte and Valle d'Aosta ALS Register between 1995 and 1996 were enrolled in the study. The patients were prospectively monitored with a standard evaluation form after diagnosis. RESULTS: Mean age at onset was 62.8 (SD = 11.2) years. According to El Escorial diagnostic criteria (EEDC), 112 patients had definite ALS, 85 probable ALS, 18 possible ALS, and six suspected ALS. The median survival time from symptom onset was 915 days (95% CI = 790 to 1065). The median survival time from diagnosis was 580 days (95% CI = 490 to 670). In univariate analysis, outcome was significantly related to age, onset site, EEDC classification, and symptom progression rate (i.e., the rate of decline of muscle strength and bulbar and respiratory function in the 6 months after diagnosis). In the Cox multivariate model, age, progression rate of respiratory, bulbar, and lower limb symptoms, EEDC classification, percutaneous endoscopic gastrostomy, and treatment with riluzole were significantly related to outcome. CONCLUSIONS: The rate of progression of symptoms in early ALS is predictive of disease outcome.

Evaluation of bioavailability of three types of IFNbeta in multiple sclerosis patients by a new quantitative-competitive-PCR method for MxA quantification.

Intracellular expression of human myxovirus protein A (MxA) is exclusively induced by type I IFNs (IFNalpha,beta,omega) or by some viruses and it is strongly increased under IFN treatment. We set up an internally controlled quantitative-competitive polymerase chain reaction (qc-PCR) that quantifies MxA mRNA expressed in human peripheral blood mononuclear cells (PBMC). Our qc-PCR is accurate because the mean ratio of copy number estimated by qc-PCR to that quantified spectrophotometrically is 1.08+/-0.03, moreover it is repeatable with high sensitivity (1 fg MxA/pg GAPDH). MxA mRNA was tested in 47 Relapsing-Remitting Multiple Sclerosis (RR-MS) untreated patients and in 48 patients treated with one of the 3 IFNbeta licensed for MS (24 with Rebif, 14 with Avonex and 10 with Betaferon). All the 48 treated patients were negative to IFNbeta neutralising antibodies (NABs) as tested in our laboratory using a cytopathic assay (CPE). MxA mRNA levels were detectable in all untreated patients (mean 24+/-18 fg MxA/pg GAPDH) and significantly higher levels were found in all the treated patients 12 h after IFNbeta administration (mean 499+/-325 fg MxA/pg GAPDH); furthermore, the three types of IFNbeta showed comparable bioavailability. Our data indicate that the bioavailability of the three available types of IFNbeta can be evaluated by MxA qc-PCR.

bcl-2 protein expression in aged brain and neurodegenerative diseases.

The proto-oncogene bcl-2 is involved in the regulation of cell death and is able to block apoptosis in neurones through reduced generation of reactive oxygen species (ROS). We have studied the immunohistochemical expression of bcl-2 protein in the aged brain and in various human neurodegenerative diseases. In all cases, bcl-2 was strongly enriched within lipofuscin and autophagic vacuoles of neurones, glial and vascular cells. Our data show that accumulation of bcl-2 is not disease-specific and represents a general cellular response which accompanies the increased formation of lipofuscin. Since oxidative stress is directly involved in lipofuscinogenesis, accumulation of bcl-2 may reflect a mechanism for counterbalancing ROS-mediated damage, or it might represent the impairment of bcl-2-dependent protection from ROS.

Glial fibrillary acidic protein and vimentin in the experimental glial reaction of the rat brain.

Glial reaction has been studied in the rat by the immunohistochemical demonstration of glial fibrillary acidic protein (GFAP) and vimentin (VIM) in two experimental conditions. The first was represented by a necrotic cerebral lesion obtained by laser irradiation and the second by the development of experimental tumors induced by transplacental ethylnitrosourea. Reactive astrocytes develop not only in the proximity of the lesion but also distant from it. The intensity of the glial response seems to depend upon the normal distribution of astrocytes and the perilesional edema. GFAP decorates all the reactive astrocytes, whereas VIM is positive only in those at the edges of the lesion. The significance of the different responses in the two models and between the two intermediate filaments is discussed.

Immunohistochemistry of glial reaction after injury in the rat: double stainings and markers of cell proliferation.

The astrocytic reaction in the rat after brain injury has been studied immunohistochemically for intermediate filaments (GFAP and vimentin), also with double staining procedures, and for markers of proliferation (BrdU and PCNA). GFAP-positive reactive astrocytes appeared around the lesion, where they were vimentin-positive and at a distance. BrdU and PCNA showed a high labelling index around the wound at day 2 and scattered positive nuclei were also found at a distance in the ipsilateral side. BrdU-positive astrocytes represented a minor fraction of GFAP- and vimentin-positive astrocytes. The expression of vimentin persisted at least 15 days after the lesion. Our results could suggest that distant reactive astrocytes originate through hypertrophy while those close to lesion arise by hyperplasia from mature or immature glial cells. The hypothesis is formulated that cells of the periventricular matrix contribute to the post-traumatic proliferative activity.

Histochemistry and electron microscopy of muscle fibres in a case of congenital paramyotonia.

In a case of congenital paramyotonia a muscle biopsy was performed and studied morphologically, histochemically and ultrastructurally. A clearcut pattern of changes has been observed with ATPase and oxidative enzymes. On electron microscopy special changes known as "tubular aggregates" were found. The relationship between the two findings, as well as the significance of such alterations in the range of periodic paralyses and myotonic phenomena, are discussed.

A syndrome resembling limbic encephalitis, associated with bronchial carcinoma, but without neuropathological abnormality: a case report.

A patient, suffering from an oat-cell bronchial carcinoma, presented with complex partial seizures, complete loss of recent memory, mild disorientation and confabulation. There was no complaint of anxiety. The rest of the neurological examination and four computed tomographic scans of the head were normal. Repeated EEG recordings were abnormal. Antiepileptic and antipsychotic treatment led to full remission within 10 days. Post-mortem examination of the brain revealed no pathological changes.

Apoptosis and cell proliferation in human neuroepithelial tumors.

Apoptosis and cell proliferation were studied in 180 human neuroepithelial tumors (30 medulloblastomas, 30 intracranial ependymomas, 30 oligodendrogliomas and 90 astrocytic tumors, including 30 astrocytomas, 30 anaplastic astrocytomas and 30 glioblastomas). Apoptotic nuclei were detected by morphology and in situ end-labeling (ISEL) of DNA breaks. The frequency of apoptotic nuclei varied from oncotype to oncotype and their distribution in each oncotype was uneven. An apoptotic index (AI) was calculated; this was high in malignant tumors and in tumors of embryonal origin and lower in tumors of the glial series. The AI/mitotic index (MI) ratio was lower in malignant tumors and higher in benign tumors, suggesting a relationship between apoptosis and cell proliferation. There was no significant correlation of either AI or AI/MI ratio with either labeling index (LI) of Ki-67 clone MIB-1 or with survival. A trends towards low AI/MI ratio in tumors with high LI and short survival was observed. Apoptosis expresses cell loss in tumors, but it did not appear to be a prognostic factor.

Clinical-anatomic study of a family with bulbo-spinal muscular atrophy in adults.

Four cases are presented, with bulbo-spinal muscular atrophy characterised by adult onset and rapid evolution. They belong to a family in which the type of inheritance is probably dominant. Two cases were studied histologically. The most striking feature was the disappearance of neurons in the lower motor nuclei of medulla and of the spinal anterior horns. An electron microscopic study was carried out in one case. Accumulation of neurofilaments was a general characteristic, in addition to the picture of different sized spheroids. The clinical-pathological relationship is discussed.

Synaptic vesicle proteins, synaptophysin and chromogranin A in amyotrophic lateral sclerosis.

In amyotrophic lateral sclerosis (ALS) it is not known which motoneuron is affected first. The study of synaptic proteins may contribute to the clarification of the problem. Fifteen cases of ALS and five control cases were studied with the immunohistochemical demonstration of synaptophysin (Sy) and chromogranin A (CgA). Sy is a typical membrane protein of small synaptic vesicles (SSV), whereas CgA is found in large dense core vesicles (LDCV) and in neurosecretory granules. In controls, Sy is distributed as dots on the neuronal surface, on proximal dendrites and in neuropil, whereas CgA is found in perikarya and dendrites and as puncta in the neuropil. In ALS there is a marked decrease of Sy-positive dots. In chromatolytic neurons and spheroids a diffuse reaction may occur. CgA-positive dots disappear in ALS, sometimes replaced by a dust-like positivity. CgA is produced by Golgi apparatus and its reduction in ALS corresponds to the fragmentation of the Golgi complex, described in the literature. The findings are interpreted as secondary to the lower motoneuron degeneration and discussed in relation to our knowledge on vesicle production and migration in the neuron and on synapses in the anterior horns of spinal cord.

Glial fibrillary acidic protein (GFAP) in rat brain tumors transplacentally induced by ethylnitrosourea (ENU).

The immunohistochemical distribution of glial fibrillary acidic protein (GFAP) in neoplastic lesions induced in the rat by ENU is reported. GFAP was present in hypertrophic reactive astrocytes, which were numerous in early neoplastic proliferations, in microtumors of the white matter, and in those collected at the periphery of large tumors. They were absent in cortical oligodendroglial foci and microtumors. No GFAP-positive cells were observed in hyperplasias of the white matter: astrocyte-like cells of large tumors were GFAP-negative. The significance of reactive astrocytes and the problem of the astrocytic component in transplacental ENU tumors are discussed.

Benign recurrence of a pilocytic cerebellar astrocytoma 36 years after radical removal: case report.

A benign recurrence of a cerebellar juvenile astrocytoma was found in a 52-year-old woman 36 years after the initial radical removal, which was performed when she was 16 years of age. To our knowledge, this is the third detailed case report of the late benign recurrence of cerebellar astrocytoma after total removal. The problem of recurrence of cerebellar astrocytoma is reviewed.