Roberto Del Bo

Identification of a Primitive Brain–Derived Neural Stem Cell Population Based on Aldehyde Dehydrogenase Activity

Stem cells are undifferentiated cells defined by their ability to self‐renew and differentiate to progenitors and terminally differentiated cells. Stem cells have been isolated from almost all tissues, and an emerging idea is that they share common characteristics such as the presence of ATP‐binding cassette transporter G2 and high telomerase and aldehyde dehydrogenase (ALDH) activity, raising the hypothesis of a set of universal stem cell markers. In the present study, we describe the isolation of primitive neural stem cells (NSCs) from adult and embryonic murine neurospheres and dissociated tissue, based on the expression of high levels of ALDH activity. Single‐cell suspension was stained with a fluorescent ALDH substrate termed Aldefluor and then analyzed by flow cytometry. A population of cells with low side scatter (SSClo) and bright ALDH (ALDHbr) activity was isolated. SSCloALDHbr cells are capable of self‐renewal and are able to generate new neurospheres and neuroepithelial stem‐like cells. Furthermore, these cells are multipotent, differentiating both in neurons and macroglia, as determined by immunocytochemistry and real‐time reverse transcription–polymerase chain reaction analysis. To evaluate the engraftment potential of SSCloALDHbr cells in vivo, we transplanted them into mouse brain. Donor‐derived neurons with mature morphology were detected in the cortex and subcortical areas, demonstrating the capacity of this cell population to differentiate appropriately in vivo. The ALDH expression assay is an effective method for direct identification of NSCs, and improvement of the stem cell isolation protocol may be useful in the development of a cell‐mediated therapeutic strategy for neurodegenerative diseases.

Reciprocal control of inflammatory cytokines, IL-1 and IL-6, and β-amyloid production in cultures ☆

To investigate the role of IL-6 in the pathogenesis of Alzheimers disease (AD) its effect on amyloid precursor protein (APP) mRNA expression was evaluated. The levels of APP mRNA were determined by Northern blot analysis in primary cultured rat cortical neurons and glial cells exposed to IL-6 (50-200 ng/ml). The cytokine increased neuronal APP mRNA expression about 100% at the highest dose after 6 h of exposure. APP mRNA expression was unaffected in astroglial cells exposed to IL-6. Since IL-1 beta also increased neuronal APP mRNA, the combination of IL-1 beta and IL-6 was tested. The effects were partially additive. The ability of beta-amyloid fragment 25-35 to induce IL-1 or IL-6 mRNA was also investigated in astroglial cells. IL-1 beta mRNA was strongly induced by beta 25-35 (25-100 microM) while the expression of IL-6 mRNA remaining unchanged. The results suggest roles for both IL-1 and IL-6 in the neuronal mechanisms related to beta-amyloid protein deposition in AD.

Mutations of FUS Gene in Sporadic Amyotrophic Lateral Sclerosis

Background Mutations in the FUS gene have recently been discovered to be a major cause of familial amyotrophic lateral sclerosis (FALS). Objective To determine the identity and frequency of FUS gene mutations in a large cohort of Italian patients enriched in sporadic cases (SALS). Methods Exons 5, 6, 14 and 15 of the FUS gene were screened for mutations in 1009 patients (45 FALS and 964 SALS). The genetic analysis was extended to the entire coding sequence of FUS in all the FALS and 293 of the SALS patients. Results Seven missense mutations (p.G191S, p.R216C, p.G225V, p.G230C, p.R234C, p.G507D and p.R521C) were identified in nine patients (seven SALS and two FALS), and none in 500 healthy Italian controls. All mutations are novel except for the p.R521C mutation identified in one SALS and one FALS case. Both patients showed a similar unusual presentation, with proximal, mostly symmetrical, upper limb weakness, with neck and axial involvement. With the exception of p.G507D and p.R521C, the mutations identified in SALS patients are all localised in the glycine-rich region encoded by exon 6. In addition, eight different in-frame deletions in two polyglycine motifs were detected, the frequency of which was not significantly different in patients and controls. Conclusions The results show that FUS missense mutations are present in 0.7% of Italian SALS cases, and confirm the previous mutational frequency reported in FALS (4.4%). An unusual proximal and axial clinical presentation seems to be associated with the presence of the p.R521C mutation.

Vascular endothelial growth factor gene variability is associated with increased risk for AD

Converging evidence points to a pivotal role of vascular endothelial growth factor (VEGF) in neuronal protection and a lack of its activity in neurodegenerative disorders. To investigate this possible association, we screened the VEGF gene promoter for various well‐known single‐nucleotide polymorphisms in a series of 249 consecutively recruited Italian patients with sporadic Alzheimers disease (AD). Genetic analysis indicated different distributions of two single‐nucleotide polymorphisms in the AD population compared with healthy control subjects. In particular, the frequencies of −2578A/A and −1198C/T genotypes were significantly greater in AD patients than in control subjects (23.7 vs 14.7% and 2.8 vs 0%, respectively). The −2578A/A genotype was associated with an increased risk for disease, independently of apolipoprotein E genotype. The risk was significantly increased with respect to various VEGF genotype combinations. In contrast, no difference in serum VEGF levels was detected comparing 96 patients and 49 control subjects. These findings suggest that polymorphisms within the promoter region of the VEGF gene confer greater risk for AD, probably by reducing its neuroprotective effect, and confirm the biological role of VEGF in neurodegenerative processes. Ann Neurol 2005;57:373–380

Neural stem cell transplantation can ameliorate the phenotype of a mouse model of spinal muscular atrophy

Spinal muscular atrophy (SMA), a motor neuron disease (MND) and one of the most common genetic causes of infant mortality, currently has no cure. Patients with SMA exhibit muscle weakness and hypotonia. Stem cell transplantation is a potential therapeutic strategy for SMA and other MNDs. In this study, we isolated spinal cord neural stem cells (NSCs) from mice expressing green fluorescent protein only in motor neurons and assessed their therapeutic effects on the phenotype of SMA mice. Intrathecally grafted NSCs migrated into the parenchyma and generated a small proportion of motor neurons. Treated SMA mice exhibited improved neuromuscular function, increased life span, and improved motor unit pathology. Global gene expression analysis of laser-capture-microdissected motor neurons from treated mice showed that the major effect of NSC transplantation was modification of the SMA phenotype toward the wild-type pattern, including changes in RNA metabolism proteins, cell cycle proteins, and actin-binding proteins. NSC transplantation positively affected the SMA disease phenotype, indicating that transplantation of NSCs may be a possible treatment for SMA.

Isolation and characterization of murine neural stem/progenitor cells based on Prominin-1 expression.

The identification of strategies for the isolation of neural stem cells (NSCs) has important implications for the understanding of their biology and the development of therapeutic applications. It has been previously described that human neural stem and progenitor cells (NSPCs) can be isolated from the central nervous system (CNS) using antibodies to prominin (CD133) and fluorescence-activated cell sorting (FACS). Although this antigen displayed an identical membrane topology in several human and murine tissues there was uncertainty as to the relationship between human and mouse prominin because of the low level of amino acid identity. Here we show that prominin expression can be used to identify and isolate also murine NSPCs from the developing or adult brain. Prominin is co-expressed with known neural stem markers like SOX 1-2, Musashi and Nestin. Moreover, neurosphere-forming cells with multipotency and self-renewal capacity reside within the prominin-positive fraction. Transplantation experiments show that CD133-positive cells give rise to neurons and glial cells in vivo, and that many neurons display appropriate phenotypic characteristics of the recipient tissues. The demonstration that CD133 is a stem cell antigen for murine NSPCs as it is for human NSPCs is useful for the investigation of mammal neurogenesis and development of preclinical tests of NSPCs transplantation in mouse analogues of human diseases.

Autosomal Dominant Frontotemporal Lobar Degeneration Due to the C9ORF72 Hexanucleotide Repeat Expansion: Late-Onset Psychotic Clinical Presentation

BACKGROUND A hexanucleotide repeat expansion in the first intron of C9ORF72 has been shown to be responsible for a high number of familial cases of amyotrophic lateral sclerosis or frontotemporal lobar degeneration (FTLD). Atypical presentations have been described, particularly psychosis. METHODS We determined the frequency of the hexanucleotide repeat expansions in a population of 651 FTLD patients and compared the clinical characteristics of carriers and noncarriers. In addition, we genotyped 21 patients with corticobasal syndrome, 31 patients with progressive supranuclear palsy, and 222 control subjects. RESULTS The pathogenic repeat expansion was detected in 39 (6%) patients with FTLD (17 male and 22 female subjects); however, it was not detected in any corticobasal syndrome and progressive supranuclear palsy patients or controls. Twenty-four of 39 carriers had positive family history for dementia and/or amyotrophic lateral sclerosis (61.5%), whereas only 145 of 612 noncarriers had positive family history (23.7%; p<.000001). Clinical phenotypes of carriers included 29 patients with the behavioral variant frontotemporal dementia (bvFTD; 5.2% of all bvFTD cases), 8 with bvFTD/motor neuron disease (32% bvFTD/motor neuron disease cases), 2 with semantic dementia (5.9% of patients with semantic dementia), and none with progressive nonfluent aphasia. The presentation with late-onset psychosis (median age = 63 years) was more frequent in carriers than noncarriers (10/33 vs. 3/37, p = .029), as well as the presence of cognitive impairment at onset (15/33 vs. 5/37; p = .0039). CONCLUSIONS The repeat expansion in C9ORF72 is a common cause of FTLD and often presents with late-onset psychosis or memory impairment.

C9ORF72 repeat expansion in a large Italian ALS cohort: evidence of a founder effect

A hexanucleotide repeat expansion (RE) in C9ORF72 gene was recently reported as the main cause of amyotrophic lateral sclerosis (ALS) and cases with frontotemporal dementia. We screened C9ORF72 in a large cohort of 259 familial ALS, 1275 sporadic ALS, and 862 control individuals of Italian descent. We found RE in 23.9% familial ALS, 5.1% sporadic ALS, and 0.2% controls. Two cases carried the RE together with mutations in other ALS-associated genes. The phenotype of RE carriers was characterized by bulbar-onset, shorter survival, and association with cognitive and behavioral impairment. Extrapyramidal and cerebellar signs were also observed in few patients. Genotype data revealed that 95% of RE carriers shared a restricted 10-single nucleotide polymorphism haplotype within the previously reported 20-single nucleotide polymorphism risk haplotype, detectable in only 27% of nonexpanded ALS cases and in 28% of controls, suggesting a common founder with cohorts of North European ancestry. Although C9ORF72 RE segregates with disease, the identification of RE both in controls and in patients carrying additional pathogenic mutations suggests that penetrance and phenotypic expression of C9ORF72 RE may depend on additional genetic risk factors.

A genome-wide association meta-analysis identifies a novel locus at 17q11.2 associated with sporadic amyotrophic lateral sclerosis

Identification of mutations at familial loci for amyotrophic lateral sclerosis (ALS) has provided novel insights into the aetiology of this rapidly progressing fatal neurodegenerative disease. However, genome-wide association studies (GWAS) of the more common (∼90%) sporadic form have been less successful with the exception of the replicated locus at 9p21.2. To identify new loci associated with disease susceptibility, we have established the largest association study in ALS to date and undertaken a GWAS meta-analytical study combining 3959 newly genotyped Italian individuals (1982 cases and 1977 controls) collected by SLAGEN (Italian Consortium for the Genetics of ALS) together with samples from Netherlands, USA, UK, Sweden, Belgium, France, Ireland and Italy collected by ALSGEN (the International Consortium on Amyotrophic Lateral Sclerosis Genetics). We analysed a total of 13 225 individuals, 6100 cases and 7125 controls for almost 7 million single-nucleotide polymorphisms (SNPs). We identified a novel locus with genome-wide significance at 17q11.2 (rs34517613 with P = 1.11 × 10(-8); OR 0.82) that was validated when combined with genotype data from a replication cohort (P = 8.62 × 10(-9); OR 0.833) of 4656 individuals. Furthermore, we confirmed the previously reported association at 9p21.2 (rs3849943 with P = 7.69 × 10(-9); OR 1.16). Finally, we estimated the contribution of common variation to heritability of sporadic ALS as ∼12% using a linear mixed model accounting for all SNPs. Our results provide an insight into the genetic structure of sporadic ALS, confirming that common variation contributes to risk and that sufficiently powered studies can identify novel susceptibility loci.

Fas small interfering RNA reduces motoneuron death in amyotrophic lateral sclerosis mice

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease characterized by selective motoneuron death. Understanding of the molecular mechanisms that trigger and regulate motoneuron degeneration could be relevant to ALS and other motoneuron disorders. This study investigates the role of Fas‐linked motoneuron death in the pathogenesis of ALS.