TREX1 p.A129fs and p.Y305C variants in a large multi-ethnic cohort of CADASIL-like unrelated patients.

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and retinal vasculopathy with cerebral leukodystrophy and systemic manifestations (RVCL-S) are the most common forms of rare monogenic early-onset cerebral small vessel disease and share clinical, and, to different extents, neuroradiological and neuropathological features. However, whether CADASIL and RVCL-S overlapping phenotype may be explained by shared genetic risk or causative factors such as TREX1 coding variants remains poorly understood. To investigate this intriguing hypothesis, we used exome sequencing to screen TREX1 protein-coding variability in a large multi-ethnic cohort of 180 early-onset independent familial and apparently sporadic CADASIL-like Caucasian patients from the USA, Portugal, Finland, Serbia and Turkey. We report 2 very rare and likely pathogenic TREX1 mutations: a loss of function mutation (p.Ala129fs) clustering in the catalytic domain, in an apparently sporadic 46-year-old patient from the USA and a missense mutation (p.Tyr305Cys) in the well conserved C-terminal region, in a 57-year-old patient with positive family history from Serbia. In concert with recent findings, our study expands the clinical spectrum of diseases associated with TREX1 mutations.

Long-Term Connectome Analysis Reveals Reshaping of Visual, Spatial Networks in a Model With Vascular Dementia Features.

BACKGROUND: Connectome analysis of neuroimaging data is a rapidly expanding field that offers the potential to diagnose, characterize, and predict neurological disease. Animal models provide insight into biological mechanisms that underpin disease, but connectivity approaches are currently lagging in the rodent. METHODS: We present a pipeline adapted for structural and functional connectivity analysis of the mouse brain, and we tested it in a mouse model of vascular dementia. RESULTS: We observed lacunar infarctions, microbleeds, and progressive white matter change across 6 months. For the first time, we report that default mode network activity is disrupted in the mouse model. We also identified specific functional circuitry that was vulnerable to vascular stress, including perturbations in a sensorimotor, visual resting state network that were accompanied by deficits in visual and spatial memory tasks. CONCLUSIONS: These findings advance our understanding of the mouse connectome and provide insight into how it can be altered by vascular insufficiency.

Shared and oppositely regulated transcriptomic signatures in Huntington's disease and brain ischemia confirm known and unveil novel potential neuroprotective genes.

Huntington's disease and subcortical vascular dementia display similar dementing features, shaped by different degrees of striatal atrophy, deep white matter degeneration and tau pathology. To investigate the hypothesis that Huntington's disease transcriptomic hallmarks may provide a window into potential protective genes upregulated during brain acute and subacute ischemia, we compared RNA sequencing signatures in the most affected brain areas of 2 widely used experimental mouse models: Huntington's disease, (R6/2, striatum and cortex and Q175, hippocampus) and brain ischemia-subcortical vascular dementia (BCCAS, striatum, cortex and hippocampus). We identified a cluster of 55 shared genes significantly differentially regulated in both models and we screened these in 2 different mouse models of Alzheimer's disease, and 96 early-onset familial and apparently sporadic small vessel ischemic disease patients. Our data support the prevalent role of transcriptional regulation upon genetic coding variability of known neuroprotective genes (Egr2, Fos, Ptgs2, Itga5, Cdkn1a, Gsn, Npas4, Btg2, Cebpb) and provide a list of potential additional ones likely implicated in different dementing disorders and worth further investigation.

PHACTR1 genetic variability is not critical in small vessel ischemic disease patients and PcomA recruitment in C57BL/6J mice.

Recently, several genome-wide association studies identified PHACTR1 as key locus for five diverse vascular disorders: coronary artery disease, migraine, fibromuscular dysplasia, cervical artery dissection and hypertension. Although these represent significant risk factors or comorbidities for ischemic stroke, PHACTR1 role in brain small vessel ischemic disease and ischemic stroke most important survival mechanism, such as the recruitment of brain collateral arteries like posterior communicating arteries (PcomAs), remains unknown. Therefore, we applied exome and genome sequencing in a multi-ethnic cohort of 180 early-onset independent familial and apparently sporadic brain small vessel ischemic disease and CADASIL-like Caucasian patients from US, Portugal, Finland, Serbia and Turkey and in 2 C57BL/6J stroke mouse models (bilateral common carotid artery stenosis [BCCAS] and middle cerebral artery occlusion [MCAO]), characterized by different degrees of PcomAs patency. We report 3 very rare coding variants in the small vessel ischemic disease-CADASIL-like cohort (p.Glu198Gln, p.Arg204Gly, p.Val251Leu) and a stop-gain mutation (p.Gln273*) in one MCAO mouse. These coding variants do not cluster in PHACTR1 known pathogenic domains and are not likely to play a critical role in small vessel ischemic disease or brain collateral circulation. We also exclude the possibility that copy number variants (CNVs) or a variant enrichment in Phactr1 may be associated with PcomA recruitment in BCCAS mice or linked to diverse vascular traits (cerebral blood flow pre-surgery, PcomA size, leptomeningeal microcollateral length and junction density during brain hypoperfusion) in C57BL/6J mice, respectively. Genetic variability in PHACTR1 is not likely to be a common susceptibility factor influencing small vessel ischemic disease in patients and PcomA recruitment in C57BL/6J mice. Nonetheless, rare variants in PHACTR1 RPEL domains may influence the stroke outcome and are worth investigating in a larger cohort of small vessel ischemic disease patients, different ischemic stroke subtypes and with functional studies.

Exploring dementia and neuronal ceroid lipofuscinosis genes in 100 FTD-like patients from 6 towns and rural villages on the Adriatic Sea cost of Apulia.

Frontotemporal dementia (FTD) refers to a complex spectrum of clinically and genetically heterogeneous disorders. Although fully penetrant mutations in several genes have been identified and can explain the pathogenic mechanisms underlying a great portion of the Mendelian forms of the disease, still a significant number of families and sporadic cases remains genetically unsolved. We performed whole exome sequencing in 100 patients with a late-onset and heterogeneous FTD-like clinical phenotype from Apulia and screened mendelian dementia and neuronal ceroid lipofuscinosis genes. We identified a nonsense mutation in SORL1 VPS domain (p.R744X), in 2 siblings displaying AD with severe language problems and primary progressive aphasia and a near splice-site mutation in CLCN6 (p.S116P) segregating with an heterogeneous phenotype, ranging from behavioural FTD to FTD with memory onset and to the logopenic variant of primary progressive aphasia in one family. Moreover 2 sporadic cases with behavioural FTD carried heterozygous mutations in the CSF1R Tyrosin kinase flanking regions (p.E573K and p.R549H). By contrast, only a minority of patients carried pathogenic C9orf72 repeat expansions (1%) and likely moderately pathogenic variants in GRN (p.C105Y, p.C389fs and p.C139R) (3%). In concert with recent studies, our findings support a common pathogenic mechanisms between FTD and neuronal ceroid lipofuscinosis and suggests that neuronal ceroid lipofuscinosis genes should be investigated also in dementia patients with predominant frontal symptoms and language impairments.

Investigating APOE, APP-Aß metabolism genes and Alzheimer's disease GWAS hits in brain small vessel ischemic disease.

Alzheimer's disease and small vessel ischemic disease frequently co-exist in the aging brain. However, pathogenic links between these 2 disorders are yet to be identified. Therefore we used Taqman genotyping, exome and RNA sequencing to investigate Alzheimer's disease known pathogenic variants and pathways: APOE ε4 allele, APP-Aß metabolism and late-onset Alzheimer's disease main genome-wide association loci (APOE, BIN1, CD33, MS4A6A, CD2AP, PICALM, CLU, CR1, EPHA1, ABCA7) in 96 early-onset small vessel ischemic disease Caucasian patients and 368 elderly neuropathologically proven controls (HEX database) and in a mouse model of cerebral hypoperfusion. Only a minority of patients (29%) carried APOE ε4 allele. We did not detect any pathogenic mutation in APP, PSEN1 and PSEN2 and report a burden of truncating mutations in APP-Aß degradation genes. The single-variant association test identified 3 common variants with a likely protective effect on small vessel ischemic disease (0.54>OR > 0.32, adj. p-value <0.05) (EPHA1 p.M900V and p.V160A and CD33 p.A14V). Moreover, 5/17 APP-Aß catabolism genes were significantly upregulated (LogFC > 1, adj. p-val<0.05) together with Apoe, Ms4a cluster and Cd33 during brain hypoperfusion and their overexpression correlated with the ischemic lesion size. Finally, the detection of Aß oligomers in the hypoperfused hippocampus supported the link between brain ischemia and Alzheimer's disease pathology.

An exploratory investigation of brain collateral circulation plasticity after cerebral ischemia in two experimental C57BL/6 mouse models.

Brain collateral circulation is an essential compensatory mechanism in response to acute brain ischemia. To study the temporal evolution of brain macro and microcollateral recruitment and their reciprocal interactions in response to different ischemic conditions, we applied a combination of complementary techniques (T2-weighted magnetic resonance imaging [MRI], time of flight [TOF] angiography [MRA], cerebral blood flow [CBF] imaging and histology) in two different mouse models. Hypoperfusion was either induced by permanent bilateral common carotid artery stenosis (BCCAS) or 60-min transient unilateral middle cerebral artery occlusion (MCAO). In both models, collateralization is a very dynamic phenomenon with a global effect affecting both hemispheres. Patency of ipsilateral posterior communicating artery (PcomA) represents the main variable survival mechanism and the main determinant of stroke lesion volume and recovery in MCAO, whereas the promptness of external carotid artery retrograde flow recruitment together with PcomA patency, critically influence survival, brain ischemic lesion volume and retinopathy in BCCAS mice. Finally, different ischemic gradients shape microcollateral density and size.

Correction: Individual and temporal variability of the retina after chronic bilateral common carotid artery occlusion (BCCAO).

[This corrects the article DOI: 10.1371/journal.pone.0193961.].

Individual and temporal variability of the retina after chronic bilateral common carotid artery occlusion (BCCAO).

Animal models of disease are an indispensable element in our quest to understand pathophysiology and develop novel therapies. Ex vivo studies have severe limitations, in particular their inability to study individual disease progression over time. In this respect, non-invasive in vivo technologies offer multiple advantages. We here used bilateral common carotid artery occlusion (BCCAO) in mice, an established model for ischemic retinopathy, and performed a multimodal in vivo and ex vivo follow-up. We used scanning laser ophthalmoscopy (SLO), ocular coherence tomography (OCT) and electroretinography (ERG) over 6 weeks followed by ex vivo analyses. BCCAO leads to vascular remodeling with thickening of veins starting at 4 weeks, loss of photoreceptor synapses with concomitant reduced b-waves in the ERG and thinning of the retina. Mononuclear phagocytes showed fluctuation of activity over time. There was large inter-individual variation in the severity of neuronal degeneration and cellular inflammatory responses. Ex vivo analysis confirmed these variable features of vascular remodeling, neurodegeneration and inflammation. In summary, we conclude that multimodal follow-up and subgroup analysis of retinal changes in BCCAO further calls into question the use of ex vivo studies with distinct single end-points. We propose that our approach can foster the understanding of retinal disease as well as the clinical translation of emerging therapeutic strategies.

Mendelian adult-onset leukodystrophy genes in Alzheimer's disease: critical influence of CSF1R and NOTCH3.

Mendelian adult-onset leukodystrophies are a spectrum of rare inherited progressive neurodegenerative disorders affecting the white matter of the central nervous system. Among these, cerebral autosomal dominant and recessive arteriopathy with subcortical infarcts and leukoencephalopathy, cerebroretinal vasculopathy, metachromatic leukodystrophy, hereditary diffuse leukoencephalopathy with spheroids, and vanishing white matter disease present with rapidly progressive dementia as dominant feature and are caused by mutations in NOTCH3, HTRA1, TREX1, ARSA, CSF1R, EIF2B1, EIF2B2, EIF2B3, EIF2B4, and EIF2B5, respectively. Given the rare incidence of these disorders and the lack of unequivocally diagnostic features, leukodystrophies are frequently misdiagnosed with common sporadic dementing diseases such as Alzheimer's disease (AD), raising the question of whether these overlapping phenotypes may be explained by shared genetic risk factors. To investigate this intriguing hypothesis, we have combined gene expression analysis (1) in 6 different AD mouse strains (APPPS1, HOTASTPM, HETASTPM, TPM, TAS10, and TAU) at 5 different developmental stages (embryo [E15], 2, 4, 8, and 18 months), (2) in APPPS1 primary cortical neurons under stress conditions (oxygen-glucose deprivation) and single-variant-based and single-gene-based (c-alpha test and sequence kernel association test (SKAT)) genetic screening in a cohort composed of 332 Caucasian late-onset AD patients and 676 Caucasian elderly controls. Csf1r was significantly overexpressed (log2FC > 1, adj. p-value < 0.05) in the cortex and hippocampus of aged HOTASTPM mice with extensive Aß dense-core plaque pathology. We identified 3 likely pathogenic mutations in CSF1R TK domain (p.L868R, p.Q691H, and p.H703Y) in our discovery and validation cohort, composed of 465 AD and mild cognitive impairment (MCI) Caucasian patients from the United Kingdom. Moreover, NOTCH3 was a significant hit in the c-alpha test (adj p-value = 0.01). Adult-onset Mendelian leukodystrophy genes are not common factors implicated in AD. Nevertheless, our study suggests a potential pathogenic link between NOTCH3, CSF1R, and sporadic late-onset AD, which warrants further investigation.

Correction to: Linkage, whole genome sequence, and biological data implicate variants in RAB10 in Alzheimer's disease resilience.

CORRECTION: The original version of this article [1] unfortunately contained a typographical error. The 'Alzheimer's Disease Neuroimaging Initiative' was erroneously included as 'Alzheimer's Disease Neuroimaging Initative' in the author list of the article.

Linkage, whole genome sequence, and biological data implicate variants in RAB10 in Alzheimer's disease resilience.

BACKGROUND: While age and the APOE ε4 allele are major risk factors for Alzheimer's disease (AD), a small percentage of individuals with these risk factors exhibit AD resilience by living well beyond 75 years of age without any clinical symptoms of cognitive decline. METHODS: We used over 200 "AD resilient" individuals and an innovative, pedigree-based approach to identify genetic variants that segregate with AD resilience. First, we performed linkage analyses in pedigrees with resilient individuals and a statistical excess of AD deaths. Second, we used whole genome sequences to identify candidate SNPs in significant linkage regions. Third, we replicated SNPs from the linkage peaks that reduced risk for AD in an independent dataset and in a gene-based test. Finally, we experimentally characterized replicated SNPs. RESULTS: Rs142787485 in RAB10 confers significant protection against AD (p value = 0.0184, odds ratio = 0.5853). Moreover, we replicated this association in an independent series of unrelated individuals (p value = 0.028, odds ratio = 0.69) and used a gene-based test to confirm a role for RAB10 variants in modifying AD risk (p value = 0.002). Experimentally, we demonstrated that knockdown of RAB10 resulted in a significant decrease in Aß42 (p value = 0.0003) and in the Aß42/Aß40 ratio (p value = 0.0001) in neuroblastoma cells. We also found that RAB10 expression is significantly elevated in human AD brains (p value = 0.04). CONCLUSIONS: Our results suggest that RAB10 could be a promising therapeutic target for AD prevention. In addition, our gene discovery approach can be expanded and adapted to other phenotypes, thus serving as a model for future efforts to identify rare variants for AD and other complex human diseases.

Clinical and genetic analyses of familial and sporadic frontotemporal dementia patients in Southern Italy.

INTRODUCTION: We investigated the clinical differences between familial and sporadic frontotemporal dementia (FTD), screening for mutations in known FTD genes. METHODS: We diagnosed 22 affected individuals belonging to eight families and 43 sporadic cases with FTD in Apulia, Southern Italy, in 2 years. Mutations in common causative FTD genes (GRN, MAPT, VCP, and TARDBP) and C9ORF72 expansions were screened. RESULTS: Behavioral variant of FTD was the most common clinical subtype (50% and 69% in familial and sporadic cases, respectively). Social conduct impairment/disinhibition, loss of insight, and inflexibility were the most frequent clinical features observed at onset. One new mutation was identified in GRN in family A. DISCUSSION: Disease onset in sporadic FTD was more frequently characterized by a clustering of behavioral symptoms with apathy and loss of personal hygiene. Mutations in common causative FTD genes are not a major cause of familial and sporadic FTD in the Southern Italian population.

A Novel Splice-Acceptor Site Mutation in GRN (c.709-2 A>T) Causes Frontotemporal Dementia Spectrum in a Large Family from Southern Italy.

Heterozygous loss of function mutations in granulin represent a significant cause of frontotemporal lobar degeneration with ubiquitin and TDP-43 inclusions (FTLD-TDP). We report a novel GRN splice site mutation (c.709-2 A>T), segregating with frontotemporal dementia spectrum in a large family from southern Italy. The GRN c.709-2 A>T is predicted to result in the skipping of exon 8, leading to non-sense mediated mRNA decay. Moreover, the PGRN plasma levels in the GRN c.709-2 A>T carriers were significantly lower (24 ng/ml) compared to controls (142.7 ng/ml) or family members non-carriers (82.0 ng/ml) (p-value = 0.005, Kruskal Wallis), suggesting progranulin haploinsufficiency. We do not report any potential pathogenic GRN mutation in a follow-up cohort composed of 6 FTD families and 43 sporadic FTD cases, from the same geographic area. Our study suggests that GRN (c.709-2 A>T) is a novel and likely very rare cause of FTD in this Italian cohort. Finally, in line with previous studies, we show that GRN haploinsufficiency leads to a heterogeneous clinical picture, and plasma progranulin levels may be a reliable tool to identify GRN loss of function mutations. However, given that a) genetic and environmental factors, gender, and age may regulate PGRN plasma levels and b) plasma progranulin levels may not reflect PGRN levels in the central nervous system, we suggest that the measurement of progranulin in the plasma should always be coupled with genetic screening of GRN for mutations.

Influence of Coding Variability in APP-Aß Metabolism Genes in Sporadic Alzheimer's Disease.

The cerebral deposition of Aß42, a neurotoxic proteolytic derivate of amyloid precursor protein (APP), is a central event in Alzheimer's disease (AD)(Amyloid hypothesis). Given the key role of APP-Aß metabolism in AD pathogenesis, we selected 29 genes involved in APP processing, Aß degradation and clearance. We then used exome and genome sequencing to investigate the single independent (single-variant association test) and cumulative (gene-based association test) effect of coding variants in these genes as potential susceptibility factors for AD, in a cohort composed of 332 sporadic and mainly late-onset AD cases and 676 elderly controls from North America and the UK. Our study shows that common coding variability in these genes does not play a major role for the disease development. In the single-variant association analysis, the main hits, none of which statistically significant after multiple testing correction (1.9e-4

ABCA7 p.G215S as potential protective factor for Alzheimer's disease.

Genome-wide association studies (GWASs) have been effective approaches to dissect common genetic variability underlying complex diseases in a systematic and unbiased way. Recently, GWASs have led to the discovery of over 20 susceptibility loci for Alzheimer's disease (AD). Despite the evidence showing the contribution of these loci to AD pathogenesis, their genetic architecture has not been extensively investigated, leaving the possibility that low frequency and rare coding variants may also occur and contribute to the risk of disease. We have used exome and genome sequencing data to analyze the single independent and joint effect of rare and low-frequency protein coding variants in 9 AD GWAS loci with the strongest effect sizes after APOE (BIN1, CLU, CR1, PICALM, MS4A6A, ABCA7, EPHA1, CD33, and CD2AP) in a cohort of 332 sporadic AD cases and 676 elderly controls of British and North-American ancestry. We identified coding variability in ABCA7 as contributing to AD risk. This locus harbors a low-frequency coding variant (p.G215S, rs72973581, minor allele frequency = 4.3%) conferring a modest but statistically significant protection against AD (p-value = 0.024, odds ratio = 0.57, 95% confidence interval = 0.41-0.80). Notably, our results are not driven by an enrichment of loss of function variants in ABCA7, recently reported as main pathogenic factor underlying AD risk at this locus. In summary, our study confirms the role of ABCA7 in AD and provides new insights that should address functional studies.

Investigating the role of rare coding variability in Mendelian dementia genes (APP, PSEN1, PSEN2, GRN, MAPT, and PRNP) in late-onset Alzheimer's disease.

The overlapping clinical and neuropathologic features between late-onset apparently sporadic Alzheimer's disease (LOAD), familial Alzheimer's disease (FAD), and other neurodegenerative dementias (frontotemporal dementia, corticobasal degeneration, progressive supranuclear palsy, and Creutzfeldt-Jakob disease) raise the question of whether shared genetic risk factors may explain the similar phenotype among these disparate disorders. To investigate this intriguing hypothesis, we analyzed rare coding variability in 6 Mendelian dementia genes (APP, PSEN1, PSEN2, GRN, MAPT, and PRNP), in 141 LOAD patients and 179 elderly controls, neuropathologically proven, from the UK. In our cohort, 14 LOAD cases (10%) and 11 controls (6%) carry at least 1 rare variant in the genes studied. We report a novel variant in PSEN1 (p.I168T) and a rare variant in PSEN2 (p.A237V), absent in controls and both likely pathogenic. Our findings support previous studies, suggesting that (1) rare coding variability in PSEN1 and PSEN2 may influence the susceptibility for LOAD and (2) GRN, MAPT, and PRNP are not major contributors to LOAD. Thus, genetic screening is pivotal for the clinical differential diagnosis of these neurodegenerative dementias.

Exome sequencing identifies 2 novel presenilin 1 mutations (p.L166V and p.S230R) in British early-onset Alzheimer's disease.

Early-onset Alzheimer's disease (EOAD) represents 1%-2% of the Alzheimer's disease (AD) cases, and it is generally characterized by a positive family history and a rapidly progressive symptomatology. Rare coding and fully penetrant variants in amyloid precursor protein (APP), presenilin 1 (PSEN1), and presenilin 2 (PSEN2) are the only causative mutations reported for autosomal dominant AD. Thus, in this study we used exome sequencing data to rapidly screen rare coding variability in APP, PSEN1, and PSEN2, in a British cohort composed of 47 unrelated EOAD cases and 179 elderly controls, neuropathologically proven. We report 2 novel and likely pathogenic variants in PSEN1 (p.L166V and p.S230R). A comprehensive catalog of rare pathogenic variants in the AD Mendelian genes is pivotal for a premortem diagnosis of autosomal dominant EOAD and for the differential diagnosis with other early onset dementias such as frontotemporal dementia (FTD) and Creutzfeldt-Jakob disease (CJD).