Modifications of the endosomal compartment in fibroblasts from sporadic Alzheimer's disease patients are associated with cognitive impairment.

Morphological alterations of the endosomal compartment have been widely described in post-mortem brains from Alzheimer's disease (AD) patients and subjects with Down syndrome (DS) who are at high risk for AD. Immunostaining with antibodies against endosomal markers such as Early Endosome Antigen 1 (EEA1) revealed increased size of EEA1-positive puncta. In DS, peripheral cells such as peripheral blood mononuclear cells (PBMCs) and fibroblasts, share similar phenotype even in the absence of AD. We previously found that PBMCs from AD patients have larger EEA1-positive puncta, correlating with brain amyloid load. Here we analysed the endosomal compartment of fibroblasts from a very well characterised cohort of AD patients (IMABio3) who underwent thorough clinical, imaging and biomarkers assessments. Twenty-one subjects were included (7 AD with mild cognitive impairment (AD-MCI), 7 AD with dementia (AD-D) and 7 controls) who had amyloid-PET at baseline (PiB) and neuropsychological tests at baseline and close to skin biopsy. Fibroblasts isolated from skin biopsies were immunostained with anti-EEA1 antibody and imaged using a spinning disk microscope. Endosomal compartment ultrastructure was also analysed by electron microscopy. All fibroblast lines were genotyped and their AD risk factors identified. Our results show a trend to an increased EEA1-positive puncta volume in fibroblasts from AD-D as compared to controls (p.adj = 0.12) and reveal enhanced endosome area in fibroblasts from AD-MCI and AD-AD versus controls. Larger puncta size correlated with PiB retention in different brain areas and with worse cognitive scores at the time of biopsy as well as faster decline from baseline to the time of biopsy. Finally, we identified three genetic risk factors for AD (ABCA1, COX7C and MYO15A) that were associated with larger EEA1 puncta volume. In conclusion, the endosomal compartment in fibroblasts could be used as cellular peripheral biomarker for both amyloid deposition and cognitive decline in AD patients.

PolyQ-expanded ataxin-3 protein levels in peripheral blood mononuclear cells correlate with clinical parameters in SCA3: a pilot study.

In view of upcoming clinical trials, quantitative molecular markers accessible in peripheral blood are of critical importance as prognostic or pharmacodynamic markers in genetic neurodegenerative diseases such as Spinocerebellar Ataxia Type 3 (SCA3), in particular for signaling target engagement. In this pilot study, we focused on the quantification of ataxin-3, the protein altered in SCA3, in human peripheral blood mononuclear cells (PBMCs) acquired from preataxic and ataxic SCA3 mutation carriers as well as healthy controls, as a molecular marker directly related to SCA3 pathophysiology. We established two different highly sensitive TR-FRET-based immunoassays to measure the protein levels of either total full-length, non-expanded and expanded, ataxin-3 or specifically polyQ-expanded ataxin-3. In PBMCs, a clear discrimination between SCA3 mutation carrier and controls were seen measuring polyQ-expanded ataxin-3 protein level. Additionally, polyQ-expanded ataxin-3 protein levels correlated with disease progression and clinical severity as assessed by the Scale for the Assessment and Rating of Ataxia. Total full-length ataxin-3 protein levels were directly influenced by the expression levels of the polyQ-expanded ataxin-3 protein, but were not correlated with clinical parameters. Assessment of ataxin-3 levels in fibroblasts or induced pluripotent stem cells allowed to distinguish mutation carriers from controls, thus providing proof-of-principle validation of our PBMC findings across cell lines. Total full-length or polyQ-expanded ataxin-3 protein was not detectable by TR-FRET assays in other biofluids like plasma or cerebrospinal fluid, indicating the need for ultra-sensitive assays for these biofluids. Standardization studies revealed that tube systems, blood sampling, and PBMC preparation may influence ataxin-3 protein levels indicating a high demand for standardized protocols in biomarker studies. In conclusion, the polyQ-expanded ataxin-3 protein is a promising candidate as a molecular target engagement marker in SCA3 in future clinical trials, determinable even in-easily accessible-peripheral blood biomaterials. These results, however, require validation in a larger cohort and further standardization of modifying conditions.

Genome-wide expression profiling and functional characterization of SCA28 lymphoblastoid cell lines reveal impairment in cell growth and activation of apoptotic pathways.

BACKGROUND: SCA28 is an autosomal dominant ataxia associated with AFG3L2 gene mutations. We performed a whole genome expression profiling using lymphoblastoid cell lines (LCLs) from four SCA28 patients and six unrelated healthy controls matched for sex and age. METHODS: Gene expression was evaluated with the Affymetrix GeneChip Human Genome U133A 2.0 Arrays and data were validated by real-time PCR. RESULTS: We found 66 genes whose expression was statistically different in SCA28 LCLs, 35 of which were up-regulated and 31 down-regulated. The differentially expressed genes were clustered in five functional categories: (1) regulation of cell proliferation; (2) regulation of programmed cell death; (3) response to oxidative stress; (4) cell adhesion, and (5) chemical homeostasis. To validate these data, we performed functional experiments that proved an impaired SCA28 LCLs growth compared to controls (p < 0.005), an increased number of cells in the G0/G1 phase (p < 0.001), and an increased mortality because of apoptosis (p < 0.05). We also showed that respiratory chain activity and reactive oxygen species levels was not altered, although lipid peroxidation in SCA28 LCLs was increased in basal conditions (p < 0.05). We did not detect mitochondrial DNA large deletions. An increase of TFAM, a crucial protein for mtDNA maintenance, and of DRP1, a key regulator of mitochondrial dynamic mechanism, suggested an alteration of fission/fusion pathways. CONCLUSIONS: Whole genome expression profiling, performed on SCA28 LCLs, allowed us to identify five altered functional categories that characterize the SCA28 LCLs phenotype, the first reported in human cells to our knowledge.

Genome-wide association study confirms BST1 and suggests a locus on 12q24 as the risk loci for Parkinson's disease in the European population.

We performed a three-stage genome-wide association study (GWAS) to identify common Parkinson's disease (PD) risk variants in the European population. The initial genome-wide scan was conducted in a French sample of 1039 cases and 1984 controls, using almost 500 000 single nucleotide polymorphisms (SNPs). Two SNPs at SNCA were found to be associated with PD at the genome-wide significance level (P < 3 × 10(-8)). An additional set of promising and new association signals was identified and submitted for immediate replication in two independent case-control studies of subjects of European descent. We first carried out an in silico replication study using GWAS data from the WTCCC2 PD study sample (1705 cases, 5200 WTCCC controls). Nominally replicated SNPs were further genotyped in a third sample of 1527 cases and 1864 controls from France and Australia. We found converging evidence of association with PD on 12q24 (rs4964469, combined P = 2.4 × 10(-7)) and confirmed the association on 4p15/BST1 (rs4698412, combined P = 1.8 × 10(-6)), previously reported in Japanese data. The 12q24 locus includes RFX4, an isoform of which, named RFX4_v3, encodes brain-specific transcription factors that regulate many genes involved in brain morphogenesis and intracellular calcium homeostasis.

Missense mutations in the AFG3L2 proteolytic domain account for ∼1.5% of European autosomal dominant cerebellar ataxias.

Spinocerebellar ataxia type 28 is an autosomal dominant form of cerebellar ataxia (ADCA) caused by mutations in AFG3L2, a gene that encodes a subunit of the mitochondrial m-AAA protease. We screened 366 primarily Caucasian ADCA families, negative for the most common triplet expansions, for point mutations in AFG3L2 using DHPLC. Whole-gene deletions were excluded in 300 of the patients, and duplications were excluded in 129 patients. We found six missense mutations in nine unrelated index cases (9/366, 2.6%): c.1961C>T (p.Thr654Ile) in exon 15, c.1996A>G (p.Met666Val), c.1997T>G (p.Met666Arg), c.1997T>C (p.Met666Thr), c.2011G>A (p.Gly671Arg), and c.2012G>A (p.Gly671Glu) in exon 16. All mutated amino acids were located in the C-terminal proteolytic domain. In available cases, we demonstrated the mutations segregated with the disease. Mutated amino acids are highly conserved, and bioinformatic analysis indicates the substitutions are likely deleterious. This investigation demonstrates that SCA28 accounts for ∼3% of ADCA Caucasian cases negative for triplet expansions and, in extenso, to ∼1.5% of all ADCA. We further confirm both the involvement of AFG3L2 gene in SCA28 and the presence of a mutational hotspot in exons 15-16. Screening for SCA28, is warranted in patients who test negative for more common SCAs and present with a slowly progressive cerebellar ataxia accompanied by oculomotor signs.

A de novo SPAST mutation leading to somatic mosaicism is associated with a later age at onset in HSP.

SPG4/SPAST, the gene-encoding spastin, is responsible for the most frequent form of autosomal dominant hereditary spastic paraplegia (HSP). SPG4-HSP is a heterogeneous disorder characterized by both interfamilial and intrafamilial variation, especially regarding the severity and the age at onset. In this study, we investigated the origin of the mutation and the factors involved in intra-familial heterogeneity in a family with a SPG4 mutation. We demonstrated that the mutation occurred de novo and show evidence of somatic mosaicism in the grandfather, who was the only affected member of six siblings. His disease began at age 55, much later than in his daughter, who had onset at age 18, and his grandson, in whom onset was at age 5. These observations indicate that de novo mutations can occur in SPG4, and that somatic mosaicism might account for intra-familial variation in SPG4-linked HSP.

Exon deletions of SPG4 are a frequent cause of hereditary spastic paraplegia.

BACKGROUND: Point mutations in SPG4, the gene encoding spastin, are a frequent cause of autosomal dominant hereditary spastic paraplegia (AD-HSP). However, standard methods for genetic analyses fail to detect exonic microdeletions. METHODS: 121 mutation-negative probands were screened for rearrangements in SPG4 by multiplex ligation-dependent probe amplification. RESULTS: 24 patients with 16 different heterozygotic exon deletions in SPG4 (20%) were identified, ranging from one exon to the whole coding sequence. Comparison with 78 patients with point mutations showed a similar clinical picture but an earlier age at onset. CONCLUSIONS: Exon deletions in SPG4 are as frequent as point mutations, and SPG4 is responsible for 40% of AD-HSP.