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1.  C9ORF72 repeat expansions in cases with previously identified pathogenic mutations 
Neurology  2013;81(15):1332-1341.
Objective:
To identify potential genetic modifiers contributing to the phenotypic variability that is detected in patients with repeat expansions in chromosome 9 open reading frame 72 (C9ORF72), we investigated the frequency of these expansions in a cohort of 334 subjects previously found to carry mutations in genes known to be associated with a spectrum of neurodegenerative diseases.
Methods:
A 2-step protocol, with a fluorescent PCR and a repeat-primed PCR, was used to determine the presence of hexanucleotide expansions in C9ORF72. For one double mutant, we performed Southern blots to assess expansion sizes, and immunohistochemistry to characterize neuropathology.
Results:
We detected C9ORF72 repeat expansions in 4 of 334 subjects (1.2% [or 1.8% of 217 families]). All these subjects had behavioral phenotypes and also harbored well-known pathogenic mutations in either progranulin (GRN: p.C466LfsX46, p.R493X, p.C31LfsX35) or microtubule-associated protein tau (MAPT: p.P301L). Southern blotting of one double mutant with a p.C466LfsX46 GRN mutation demonstrated a long repeat expansion in brain (>3,000 repeats), and immunohistochemistry showed mixed neuropathology with characteristics of both C9ORF72 expansions and GRN mutations.
Conclusions:
Our findings indicate that co-occurrence of 2 evidently pathogenic mutations could contribute to the pleiotropy that is detected in patients with C9ORF72 repeat expansions. These findings suggest that patients with known mutations should not be excluded from further studies, and that genetic counselors should be aware of this phenomenon when advising patients and their family members.
doi:10.1212/WNL.0b013e3182a8250c
PMCID: PMC3806926  PMID: 24027057
2.  New Genes and New Insights from Old Genes: Update on Alzheimer Disease 
Continuum : Lifelong Learning in Neurology  2013;19(2 Dementia):358-371.
Purpose of Review: This article discusses the current status of knowledge regarding the genetic basis of Alzheimer disease (AD) with a focus on clinically relevant aspects.
Recent Findings: The genetic architecture of AD is complex, as it includes multiple susceptibility genes and likely nongenetic factors. Rare but highly penetrant autosomal dominant mutations explain a small minority of the cases but have allowed tremendous advances in understanding disease pathogenesis. The identification of a strong genetic risk factor, APOE, reshaped the field and introduced the notion of genetic risk for AD. More recently, large-scale genome-wide association studies are adding to the picture a number of common variants with very small effect sizes. Large-scale resequencing studies are expected to identify additional risk factors, including rare susceptibility variants and structural variation.
Summary: Genetic assessment is currently of limited utility in clinical practice because of the low frequency (Mendelian mutations) or small effect size (common risk factors) of the currently known susceptibility genes. However, genetic studies are identifying with confidence a number of novel risk genes, and this will further our understanding of disease biology and possibly the identification of therapeutic targets.
doi:10.1212/01.CON.0000429179.21977.a1
PMCID: PMC3915548  PMID: 23558482
3.  Greater medial temporal hypometabolism and lower cortical amyloid burden in ApoE4-positive AD patients 
Background
ApoE4 has been associated with an increased risk of Alzheimer’s disease (AD), amyloid deposition and hypometabolism. ApoE4 is less prevalent in non-amnestic AD variants suggesting a direct effect on the clinical phenotype. However, the impact of ApoE4 on amyloid burden and glucose metabolism across different clinical AD syndromes is not well understood. We aimed to assess the relationship between amyloid deposition, glucose metabolism and ApoE4 genotype in a clinically heterogeneous population of AD patients.
Methods
Fifty-two patients with probable AD (NIA-AA) underwent [11C]Pittsburgh compound B (PIB) and [18F]fluorodeoxyglucose (FDG) PET scans. All patients had positive PIB-PET scans. 23 were ApoE4+ (14 heterozygous, 9 homozygous) and 29 were ApoE4−. Groups consisted of language-variant AD, visual-variant AD, and AD patients with amnestic and dysexecutive deficits. 52 healthy controls were included for comparison. FDG and PIB uptake was compared between groups on a voxel-wise basis and in regions-of-interest.
Results
Whilst PIB patterns were diffuse in both patient groups, ApoE4− patients showed higher PIB uptake than ApoE4+ patients across the cortex. Higher PIB uptake in ApoE4− patients was particularly significant in right lateral frontotemporal regions. In contrast, similar patterns of hypometabolism relative to controls were found in both patient groups, mainly involving lateral temporoparietal cortex, precuneus, posterior cingulate cortex, and middle frontal gyrus. Comparing patient groups, ApoE4+ subjects showed greater hypometabolism in bilateral medial temporal and right lateral temporal regions, and ApoE4− patients showed greater hypometabolism in cortical areas including supplementary motor cortex and superior frontal gyrus.
Conclusions
ApoE4+ AD patients showed lower global amyloid burden and greater medial temporal hypometabolism compared to matched ApoE4− patients. These findings suggest that ApoE4 may increase susceptibility to molecular pathology and modulate the anatomic pattern of neurodegeneration in AD.
doi:10.1136/jnnp-2013-305858
PMCID: PMC3946299  PMID: 23965289
Alzheimer’s disease; PET; amyloid; glucose metabolism; apolipoprotein E
4.  An Epigenetic Signature in Peripheral Blood Associated with the Haplotype on 17q21.31, a Risk Factor for Neurodegenerative Tauopathy 
PLoS Genetics  2014;10(3):e1004211.
Little is known about how changes in DNA methylation mediate risk for human diseases including dementia. Analysis of genome-wide methylation patterns in patients with two forms of tau-related dementia – progressive supranuclear palsy (PSP) and frontotemporal dementia (FTD) – revealed significant differentially methylated probes (DMPs) in patients versus unaffected controls. Remarkably, DMPs in PSP were clustered within the 17q21.31 region, previously known to harbor the major genetic risk factor for PSP. We identified and replicated a dose-dependent effect of the risk-associated H1 haplotype on methylation levels within the region in blood and brain. These data reveal that the H1 haplotype increases risk for tauopathy via differential methylation at that locus, indicating a mediating role for methylation in dementia pathophysiology.
Author Summary
Progressive supranuclear palsy (PSP) and frontotemporal dementia (FTD) are two neurodegenerative diseases linked, at the pathologic and genetic level, to the microtubule associated protein tau. We studied epigenetic changes (DNA methylation levels) in peripheral blood from patients with PSP, FTD, and unaffected controls. Analysis of genome-wide methylation patterns revealed significant differentially methylated probes in patients versus unaffected controls. Remarkably, differentially methylated probes in PSP vs. controls were preferentially clustered within the 17q21.31 region, previously known to harbor the major genetic risk factor for PSP. We identified and replicated a dose-dependent effect of the risk-associated H1 haplotype on methylation levels within the region in independent datasets in blood and brain. These data reveal that the H1 haplotype increases risk for tauopathy via differential methylation, indicating a mediating role for methylation in dementia pathophysiology.
doi:10.1371/journal.pgen.1004211
PMCID: PMC3945475  PMID: 24603599
6.  Mutations in PDYN are Not Responsible for Multiple System Atrophy 
Journal of neurology  2013;260(3):927-928.
doi:10.1007/s00415-012-6830-x
PMCID: PMC3594076  PMID: 23355175
ataxia; cerebellum; PDYN; SCA23; MSA; multiple system atrophy
7.  The effect of the serotonin transporter (5-HTTLPR) polymorphism on empathic and self-conscious emotional reactivity 
Emotion (Washington, D.C.)  2012;13(1):25-35.
We examined the relationship between a functional polymorphism of the serotonin transporter (5-HTTLPR) gene and individual differences in emotional reactivity in two laboratory studies. In study 1, empathic responding and physiological reactivity to viewing films of others in distress were assessed in healthy adults in three age groups. In study 2, emotional responding to watching oneself in an embarrassing situation was assessed in healthy adults and in patients with neurodegenerative diseases. In study 1, participants with two short alleles of 5-HTTLPR reported more personal distress and showed higher levels of physiological responses in response to the films than participants with long alleles. In study 2, participants with two short alleles reported more anger and amusement and displayed more emotional expressive behaviors in response to the embarrassing situation than participants with long alleles. These two findings from diverse samples of participants converge to indicate that individuals who are homozygous for the short allele variant of 5-HTTLPR have greater levels of emotional reactivity in two quite different socially-embedded contexts.
doi:10.1037/a0029616
PMCID: PMC3553251  PMID: 22906085
5-HTTLPR; empathy; self-conscious emotions; physiology; reactivity
8.  Regional brain volume differences in symptomatic and presymptomatic carriers of familial Alzheimer’s disease mutations 
Background
Mutations in the presenilin (PSEN1, PSEN2) and amyloid precursor protein (APP) genes cause familial Alzheimer’s disease (FAD) in a nearly fully penetrant, autosomal dominant manner, providing a unique opportunity to study presymptomatic individuals who can be predicted to develop Alzheimer’s disease (AD) with essentially 100% certainty. Using tensor-based morphometry (TBM), we examined brain volume differences between presymptomatic and symptomatic FAD mutation carriers and non-carrier (NC) relatives.
Methods
Twenty-five mutation carriers and 10 NC relatives underwent brain MRI and clinical assessment. Four mutation carriers had dementia (MUT-Dem), 12 had amnestic mild cognitive impairment (MUT-aMCI) and nine were cognitively normal (MUT-Norm). TBM brain volume maps of MUT-Norm, MUT-aMCI and MUT-Dem subjects were compared to NC subjects.
Results
MUT-Norm subjects exhibited significantly smaller volumes in the thalamus, caudate and putamen. MUT-aMCI subjects had smaller volumes in the thalamus, splenium and pons, but not in the caudate or putamen. MUT-Dem subjects demonstrated smaller volumes in temporal, parietal and left frontal regions. As non-demented carriers approached the expected age of dementia diagnosis, this was associated with larger ventricular and caudate volumes and a trend towards smaller temporal lobe volume.
Conclusions
Cognitively intact FAD mutation carriers had lower thalamic, caudate and putamen volumes, and we found preliminary evidence for increasing caudate size during the predementia stage. These regions may be affected earliest during prodromal stages of FAD, while cortical atrophy may occur in later stages, when carriers show cognitive deficits. Further studies of this population will help us understand the progression of neurobiological changes in AD.
doi:10.1136/jnnp-2011-302087
PMCID: PMC3779052  PMID: 23085935
10.  A Shift to Organismal Stress Resistance in Programmed Cell Death Mutants 
PLoS Genetics  2013;9(9):e1003714.
Animals have many ways of protecting themselves against stress; for example, they can induce animal-wide, stress-protective pathways and they can kill damaged cells via apoptosis. We have discovered an unexpected regulatory relationship between these two types of stress responses. We find that C. elegans mutations blocking the normal course of programmed cell death and clearance confer animal-wide resistance to a specific set of environmental stressors; namely, ER, heat and osmotic stress. Remarkably, this pattern of stress resistance is induced by mutations that affect cell death in different ways, including ced-3 (cell death defective) mutations, which block programmed cell death, ced-1 and ced-2 mutations, which prevent the engulfment of dying cells, and progranulin (pgrn-1) mutations, which accelerate the clearance of apoptotic cells. Stress resistance conferred by ced and pgrn-1 mutations is not additive and these mutants share altered patterns of gene expression, suggesting that they may act within the same pathway to achieve stress resistance. Together, our findings demonstrate that programmed cell death effectors influence the degree to which C. elegans tolerates environmental stress. While the mechanism is not entirely clear, it is intriguing that animals lacking the ability to efficiently and correctly remove dying cells should switch to a more global animal-wide system of stress resistance.
Author Summary
As an animal interacts with its environment, it invariably encounters stressful conditions such as extreme temperatures, drought, UV exposure and harmful xenobiotics. Since the ability to respond appropriately to stressful stimuli is paramount to survival, organisms have developed sophisticated stress response programs. Some stressful conditions cause damaged cells to commit suicide (undergo apoptosis), whereas others cause the entire organism to develop mechanisms to resist environmental stress. Studying the small roundworm C. elegans, we find that these two responses are somehow linked: perturbing the mechanisms that allow cells to undergo apoptosis changes the whole animal's response to environmental stress. In fact, perturbing the apoptosis machinery in any way—through mutations that prevent apoptosis altogether, or through mutations that either slow or accelerate the clearance of dying cells—causes the animal to become more stress resistant. Together our findings raise the possibility that the animal may have a way of detecting defects in the normal programmed cell death pathway, and that in response it induces a new program that protects itself from a harsh environment.
doi:10.1371/journal.pgen.1003714
PMCID: PMC3778000  PMID: 24068943
11.  Sumoylated MEF2A Coordinately Eliminates Orphan Presynaptic Sites and Promotes Maturation of Presynaptic Boutons 
Presynaptic differentiation of axons plays a fundamental role in the establishment of neuronal connectivity. However, the mechanisms that govern presynaptic differentiation in the brain remain largely to be elucidated. We report that knockdown of the transcription factor MEF2A in primary neurons and importantly in the rat cerebellar cortex in vivo robustly increases the density of orphan presynaptic sites. Remarkably, the sumoylated transcriptional repressor form of MEF2A drives the suppression of orphan presynaptic sites. We also identify the gene encoding synaptotagmin 1 (Syt1), which acts locally at presynaptic sites, as a direct repressed target gene of sumoylated MEF2A in neurons, and demonstrate that repression of Syt1 mediates MEF2A-dependent elimination of orphan presynaptic sites. Finally, we uncover a role for the MEF2A-induced elimination of orphan presynaptic sites in the accumulation of presynaptic material at large maturing presynaptic boutons. Collectively, these findings define sumoylated MEF2A and Syt1 as components of a novel cell-intrinsic mechanism that orchestrates presynaptic differentiation in the mammalian brain. Our study has important implications for understanding neuronal connectivity in brain development and disease.
doi:10.1523/JNEUROSCI.4191-12.2013
PMCID: PMC3740195  PMID: 23486945
12.  Frontotemporal dementia due to C9ORF72 mutations 
Neurology  2012;79(10):1002-1011.
Objective:
To describe the phenotype of patients with C9FTD/ALS (C9ORF72) hexanucleotide repeat expansion.
Methods:
A total of 648 patients with frontotemporal dementia (FTD)–related clinical diagnoses and Alzheimer disease (AD) dementia were tested for C9ORF72 expansion and 31 carried expanded repeats (C9+). Clinical and neuroimaging data were compared between C9+ (15 behavioral variant FTD [bvFTD], 11 FTD–motor neuron disease [MND], 5 amyotrophic lateral sclerosis [ALS]) and sporadic noncarriers (48 bvFTD, 19 FTD-MND, 6 ALS).
Results:
All C9+ patients displayed clinical syndromes of bvFTD, ALS, or FTD-MND. At first evaluation, C9+ bvFTD patients had more delusions and greater impairment of working memory, but milder eating dysregulation compared to bvFTD noncarriers. C9+FTD-MND patients had a trend for longer survival and had an earlier age at onset than FTD-MND noncarriers. Voxel-based morphometry demonstrated more thalamic atrophy in FTD and FTD-MND carriers than in noncarriers.
Conclusions:
Patients with the C9ORF72 hexanucleotide repeat expansion develop bvFTD, ALS, or FTD-MND with similar clinical and imaging features to sporadic cases. Other FTD spectrum diagnoses and AD dementia appear rare or absent among C9+ individuals. Longer survival in C9+ FTD-MND suggests slower disease progression and thalamic atrophy represents a novel and unexpected feature.
doi:10.1212/WNL.0b013e318268452e
PMCID: PMC3430713  PMID: 22875087
13.  The Disruption of Celf6, a Gene Identified by Translational Profiling of Serotonergic Neurons, Results in Autism-Related Behaviors 
The immense molecular diversity of neurons challenges our ability to understand the genetic and cellular etiology of neuropsychiatric disorders. Leveraging knowledge from neurobiology may help parse the genetic complexity: identifying genes important for a circuit that mediates a particular symptom of a disease may help identify polymorphisms that contribute to risk for the disease as a whole. The serotonergic system has long been suspected in disorders that have symptoms of repetitive behaviors and resistance to change, including autism. We generated a bacTRAP mouse line to permit translational profiling of serotonergic neurons. From this, we identified several thousand serotonergic-cell expressed transcripts, of which 174 were highly enriched, including all known markers of these cells. Analysis of common variants near the corresponding genes in the AGRE collection implicated the RNA binding protein CELF6 in autism risk. Screening for rare variants in CELF6 identified an inherited premature stop codon in one of the probands. Subsequent disruption of Celf6 in mice resulted in animals exhibiting resistance to change and decreased ultrasonic vocalization as well as abnormal levels of serotonin in the brain. This work provides a reproducible and accurate method to profile serotonergic neurons under a variety of conditions and suggests a novel paradigm for gaining information on the etiology of psychiatric disorders.
doi:10.1523/JNEUROSCI.4762-12.2013
PMCID: PMC3711589  PMID: 23407934
14.  Evidence for a role of the rare p.A152T variant in MAPT in increasing the risk for FTD-spectrum and Alzheimer's diseases 
Coppola, Giovanni | Chinnathambi, Subashchandrabose | Lee, Jason JiYong | Dombroski, Beth A. | Baker, Matt C. | Soto-Ortolaza, Alexandra I. | Lee, Suzee E. | Klein, Eric | Huang, Alden Y. | Sears, Renee | Lane, Jessica R. | Karydas, Anna M. | Kenet, Robert O. | Biernat, Jacek | Wang, Li-San | Cotman, Carl W. | DeCarli, Charles S. | Levey, Allan I. | Ringman, John M. | Mendez, Mario F. | Chui, Helena C. | Le Ber, Isabelle | Brice, Alexis | Lupton, Michelle K. | Preza, Elisavet | Lovestone, Simon | Powell, John | Graff-Radford, Neill | Petersen, Ronald C. | Boeve, Bradley F. | Lippa, Carol F. | Bigio, Eileen H. | Mackenzie, Ian | Finger, Elizabeth | Kertesz, Andrew | Caselli, Richard J. | Gearing, Marla | Juncos, Jorge L. | Ghetti, Bernardino | Spina, Salvatore | Bordelon, Yvette M. | Tourtellotte, Wallace W. | Frosch, Matthew P. | Vonsattel, Jean Paul G. | Zarow, Chris | Beach, Thomas G. | Albin, Roger L. | Lieberman, Andrew P. | Lee, Virginia M. | Trojanowski, John Q. | Van Deerlin, Vivianna M. | Bird, Thomas D. | Galasko, Douglas R. | Masliah, Eliezer | White, Charles L. | Troncoso, Juan C. | Hannequin, Didier | Boxer, Adam L. | Geschwind, Michael D. | Kumar, Satish | Mandelkow, Eva-Maria | Wszolek, Zbigniew K. | Uitti, Ryan J. | Dickson, Dennis W. | Haines, Jonathan L. | Mayeux, Richard | Pericak-Vance, Margaret A. | Farrer, Lindsay A. | Ross, Owen A. | Rademakers, Rosa | Schellenberg, Gerard D. | Miller, Bruce L. | Mandelkow, Eckhard | Geschwind, Daniel H.
Human Molecular Genetics  2012;21(15):3500-3512.
Rare mutations in the gene encoding for tau (MAPT, microtubule-associated protein tau) cause frontotemporal dementia-spectrum (FTD-s) disorders, including FTD, progressive supranuclear palsy (PSP) and corticobasal syndrome, and a common extended haplotype spanning across the MAPT locus is associated with increased risk of PSP and Parkinson's disease. We identified a rare tau variant (p.A152T) in a patient with a clinical diagnosis of PSP and assessed its frequency in multiple independent series of patients with neurodegenerative conditions and controls, in a total of 15 369 subjects.
Tau p.A152T significantly increases the risk for both FTD-s (n = 2139, OR = 3.0, CI: 1.6–5.6, P = 0.0005) and Alzheimer's disease (AD) (n = 3345, OR = 2.3, CI: 1.3–4.2, P = 0.004) compared with 9047 controls. Functionally, p.A152T (i) decreases the binding of tau to microtubules and therefore promotes microtubule assembly less efficiently; and (ii) reduces the tendency to form abnormal fibers. However, there is a pronounced increase in the formation of tau oligomers. Importantly, these findings suggest that other regions of the tau protein may be crucial in regulating normal function, as the p.A152 residue is distal to the domains considered responsible for microtubule interactions or aggregation. These data provide both the first genetic evidence and functional studies supporting the role of MAPT p.A152T as a rare risk factor for both FTD-s and AD and the concept that rare variants can increase the risk for relatively common, complex neurodegenerative diseases, but since no clear significance threshold for rare genetic variation has been established, some caution is warranted until the findings are further replicated.
doi:10.1093/hmg/dds161
PMCID: PMC3392107  PMID: 22556362
15.  Subcellular Knockout of Importin β1 Perturbs Axonal Retrograde Signaling 
Neuron  2012;75(2):294-305.
Summary
Subcellular localization of mRNA enables compartmentalized regulation within large cells. Neurons are the longest known cells, however so far evidence is lacking for an essential role of endogenous mRNA localization in axons. Localized upregulation of importin β1 in lesioned axons coordinates a retrograde injury signaling complex transported to the neuronal cell body. Here we show that a long 3′ untranslated region (3′UTR) directs axonal localization of importin β1. Conditional targeting of this 3′UTR region in mice causes subcellular loss of importin β1 mRNA and protein in axons, without affecting cell body levels or nuclear functions in sensory neurons. Strikingly, axonal knockout of importin β1 attenuates cell body transcriptional responses to nerve injury and delays functional recovery in vivo. Thus, localized translation of importin β1 mRNA enables separation of cytoplasmic and nuclear transport functions of importins, and is required for efficient retrograde signaling in injured axons.
doi:10.1016/j.neuron.2012.05.033
PMCID: PMC3408616  PMID: 22841314
16.  Mithramycin Is a Gene-Selective Sp1 Inhibitor That Identifies a Biological Intersection between Cancer and Neurodegeneration 
Oncogenic transformation of postmitotic neurons triggers cell death, but the identity of genes critical for degeneration remain unclear. The antitumor antibiotic mithramycin prolongs survival of mouse models of Huntington’s disease in vivo and inhibits oxidative stress-induced death in cortical neurons in vitro. We had correlated protection by mithramycin with its ability to bind to GC-rich DNA and globally displace Sp1 family transcription factors. To understand how antitumor drugs prevent neurodegeneration, here we use structure-activity relationships of mithramycin analogs to discover that selective DNA-binding inhibition of the drug is necessary for its neuroprotective effect. We identify several genes (Myc, c-Src, Hif1α, and p21waf1/cip1) involved in neoplastic transformation, whose altered expression correlates with protective doses of mithramycin or its analogs. Most interestingly, inhibition of one these genes, Myc, is neuroprotective, whereas forced expression of Myc induces Rattus norvegicus neuronal cell death. These results support a model in which cancer cell transformation shares key genetic components with neurodegeneration.
doi:10.1523/JNEUROSCI.0710-11.2011
PMCID: PMC3717375  PMID: 21543616
17.  Network Organization of the Huntingtin Proteomic Interactome in Mammalian Brain 
Neuron  2012;75(1):41-57.
SUMMARY
We used affinity-purification mass spectrometry to identify 747 candidate proteins that are complexed with Huntingtin (Htt) in distinct brain regions and ages in Huntington’s disease (HD) and wildtype mouse brains. To gain a systems-level view of the Htt interactome, we applied Weighted Gene Correlation Network Analysis (WGCNA) to the entire proteomic dataset to unveil a verifiable rank of Htt-correlated proteins and a network of Htt-interacting protein modules, with each module highlighting distinct aspects of Htt biology. Importantly, the Htt-containing module is highly enriched with proteins involved in 14-3-3 signaling, microtubule-based transport, and proteostasis. Top-ranked proteins in this module were validated as novel Htt interactors and genetic modifiers in an HD Drosophila model. Together, our study provides a compendium of spatiotemporal Htt-interacting proteins in the mammalian brain, and presents a conceptually novel approach to analyze proteomic interactome datasets to build in vivo protein networks in complex tissues such as the brain.
doi:10.1016/j.neuron.2012.05.024
PMCID: PMC3432264  PMID: 22794259
18.  Plasma methionine sulfoxide in persons with familial Alzheimer’s disease mutations 
Background
Convergent evidence suggests that oxidative stress plays a central role in the pathology of Alzheimer’s disease (AD). We asked if consequently, oxidation of methionine residues to methionine sulfoxide (MetO) increased in plasma proteins of persons carrying familial AD (FAD) mutations.
Methods
Plasma was collected from 31 persons from families harboring PSEN1 or APP mutations. Using Western blot analysis with a novel anti-MetO polyclonal antibody, MetO levels were measured and compared between FAD mutation carriers (MCs) and non-mutation carrying (NCs) kin.
Results
A MetO-positive 120 kDa gel band distinguished FAD MCs and NCs (mean 11.4 ± 2.8 vs. 4.0 ± 3.1, p = 0.02). In a subset of subjects for whom both measurements were available, MetO levels correlated well with plasma F2-isoprostane (r = 0.81, p < 0.001) and superoxide dismutase 1 (r = 0.52, p = 0.004) levels.
Conclusion
Our data provide evidence for elevated MetO levels in persons carrying FAD mutations that correlate with other indices of oxidative stress and suggest that plasma oxidative stress markers may be useful for diagnosis of AD.
doi:10.1159/000338546
PMCID: PMC3568669  PMID: 22584618
oxidative stress; isoprostanes; plasma; superoxide dismutase; presenilin-1; amyloid β-protein precursor
19.  CONDITIONING LESIONS BEFORE OR AFTER SPINAL CORD INJURY RECRUIT BROAD GENETIC MECHANISMS THAT SUSTAIN AXONAL REGENERATION: SUPERIORITY TO CAMP-MEDIATED EFFECTS 
Experimental Neurology  2011;235(1):162-173.
Previous studies indicate that peripheral nerve conditioning lesions significantly enhance central axonal regeneration via modulation of cAMP-mediated mechanisms. To gain insight into the nature and temporal dependence of neural mechanisms underlying conditioning lesion effects on central axonal regeneration, we compared the efficacy of peripheral sciatic nerve crush lesions to cAMP elevations (in lumbar dorsal root ganglia) on central sensory axonal regeneration when administered either before or after cervical spinal cord lesions. We found significantly greater effects of conditioning lesions compared to cAMP elevations on central axonal regeneration when combined with cellular grafts at the lesion site and viral neurotrophin delivery; further, these effects persisted whether conditioning lesions were applied prior to or shortly after spinal cord injury. Indeed, conditioning lesions recruited extensively greater sets of genetic mechanisms of possible relevance to axonal regeneration compared to cAMP administration, and sustained these changes for significantly greater time periods through the post-lesion period. We conclude that cAMP-mediated mechanisms account for only a portion of the potency of conditioning lesions on central axonal regeneration, and that recruitment of broader genetic mechanisms can extend the effect and duration of cellular events that support axonal growth.
doi:10.1016/j.expneurol.2011.12.037
PMCID: PMC3334479  PMID: 22227059
neurotrophin-3; CTB; regeneration; spinal cord injury; microarray; dorsal column sensory; lentivirus
20.  Microarray and Pathway Analysis Reveal Distinct Mechanisms Underlying Cannabinoid-Mediated Modulation of LPS-Induced Activation of BV-2 Microglial Cells 
PLoS ONE  2013;8(4):e61462.
Cannabinoids are known to exert immunosuppressive activities. However, the mechanisms which contribute to these effects are unknown. Using lipopolysaccharide (LPS) to activate BV-2 microglial cells, we examined how Δ9-tetrahydrocannabinol (THC), the major psychoactive component of marijuana, and cannabidiol (CBD) the non-psychoactive component, modulate the inflammatory response. Microarray analysis of genome-wide mRNA levels was performed using Illumina platform and the resulting expression patterns analyzed using the Ingenuity Pathway Analysis to identify functional subsets of genes, and the Ingenuity System Database to denote the gene networks regulated by CBD and THC. From the 5338 transcripts that were differentially expressed across treatments, 400 transcripts were found to be upregulated by LPS, 502 by CBD+LPS and 424 by THC+LPS, while 145 were downregulated by LPS, 297 by CBD+LPS and 149 by THC+LPS, by 2-fold or more (p≤0.005). Results clearly link the effects of CBD and THC to inflammatory signaling pathways and identify new cannabinoid targets in the MAPK pathway (Dusp1, Dusp8, Dusp2), cell cycle related (Cdkn2b, Gadd45a) as well as JAK/STAT regulatory molecules (Socs3, Cish, Stat1). The impact of CBD on LPS-stimulated gene expression was greater than that of THC. We attribute this difference to the fact that CBD highly upregulated several genes encoding negative regulators of both NFκB and AP-1 transcriptional activities, such as Trib3 and Dusp1 known to be modulated through Nrf2 activation. The CBD-specific expression profile reflected changes associated with oxidative stress and glutathione depletion via Trib3 and expression of ATF4 target genes. Furthermore, the CBD affected genes were shown to be controlled by nuclear factors usually involved in regulation of stress response and inflammation, mainly via Nrf2/Hmox1 axis and the Nrf2/ATF4-Trib3 pathway. These observations indicate that CBD, and less so THC, induce a cellular stress response and that this response underlies their high immunosuppressant activities.
doi:10.1371/journal.pone.0061462
PMCID: PMC3634783  PMID: 23637839
21.  Functional Genomic Analyses Identify Pathways Dysregulated by Progranulin Deficiency Implicating Wnt Signaling 
Neuron  2011;71(6):1030-1042.
Summary
Progranulin (GRN) mutations cause frontotemporal dementia (FTD), but GRN’s function in the CNS remains largely unknown. To identify the pathways downstream of GRN, we used weighted gene co-expression network analysis (WGCNA) to develop a systems-level view of transcriptional alterations in a human neural progenitor model of GRN-deficiency. This highlighted key pathways such as apoptosis and ubiquitination in GRN deficient human neurons, while revealing an unexpected major role for the Wnt signaling pathway, which was confirmed by analysis of gene expression data from postmortem FTD brain. Furthermore, we observed that the Wnt receptor Fzd2 was one of only a few genes up-regulated at 6 weeks in a GRN knockout mouse, and that FZD2 reduction caused increased apoptosis, while its upregulation promoted neuronal survival in vitro. Together, these in vitro and in vivo data point to an adaptive role for altered Wnt signaling in GRN deficiency-mediated FTD, representing a potential therapeutic target.
doi:10.1016/j.neuron.2011.07.021
PMCID: PMC3633414  PMID: 21943601
Progranulin; Frontotemporal Dementia; Wnt; Fzd2; WGCNA
22.  Inflammatory Mediators Alter the Astrocyte Transcriptome and Calcium Signaling Elicited by Multiple G-Protein-Coupled Receptors 
Inflammation features in CNS disorders such as stroke, trauma, neurodegeneration, infection, and autoimmunity in which astrocytes play critical roles. To elucidate how inflammatory mediators alter astrocyte functions, we examined effects of transforming growth factor-β1 (TGF-β1), lipopolysaccharide (LPS), and interferon-gamma (IFNγ), alone and in combination, on purified, mouse primary cortical astrocyte cultures. We used microarrays to conduct whole-genome expression profiling, and measured calcium signaling, which is implicated in mediating dynamic astrocyte functions. Combinatorial exposure to TGF-β1, LPS, and IFNγ significantly modulated astrocyte expression of >6800 gene probes, including >380 synergistic changes not predicted by summing individual treatment effects. Bioinformatic analyses revealed significantly and markedly upregulated molecular networks and pathways associated in particular with immune signaling and regulation of cell injury, death, growth, and proliferation. Highly regulated genes included chemokines, growth factors, enzymes, channels, transporters, and intercellular and intracellular signal transducers. Notably, numerous genes for G-protein-coupled receptors (GPCRs) and G-protein effectors involved in calcium signaling were significantly regulated, mostly down (for example, Cxcr4, Adra2a, Ednra, P2y1, Gnao1, Gng7), but some up (for example, P2y14, P2y6, Ccrl2, Gnb4). We tested selected cases and found that changes in GPCR gene expression were accompanied by significant, parallel changes in astrocyte calcium signaling evoked by corresponding GPCR-specific ligands. These findings identify pronounced changes in the astrocyte transcriptome induced by TGF-β1, LPS, and IFNγ, and show that these inflammatory stimuli upregulate astrocyte molecular networks associated with immune- and injury-related functions and significantly alter astrocyte calcium signaling stimulated by multiple GPCRs.
doi:10.1523/JNEUROSCI.1256-12.2012
PMCID: PMC3518872  PMID: 23077035
23.  Mutations In Rare Ataxia Genes Are Uncommon Causes of Sporadic Cerebellar Ataxia 
Movement Disorders  2012;27(3):442-446.
BACKGROUND
Sporadic-onset ataxia is common in a tertiary care setting but a significant percentage remains unidentified despite extensive evaluation. Rare genetic ataxias, reported only in specific populations or families, may contribute to a percentage of sporadic ataxia.
METHODS
Patients with adult-onset sporadic ataxia, who tested negative for common genetic ataxias (SCA1, SCA2, SCA3, SCA6, SCA7, and/or Friedreich ataxia), were evaluated using a stratified screening approach for variants in seven rare ataxia genes.
RESULTS
We screened patients for published mutations in SYNE1 (n=80) and TGM6 (n=118), copy number variations in LMNB1 (n=40) and SETX (n=11), sequence variants in SACS (n=39) and PDYN (n=119), and the pentanucleotide insertion of spinocerebellar ataxia type 31 (n=101). Overall, we identified one patient with a LMNB1 duplication, one patient with a PDYN variant, and one compound SACS heterozygote, including a novel variant.
CONCLUSIONS
The rare genetic ataxias examined here do not significantly contribute to sporadic cerebellar ataxia in our tertiary care population.
doi:10.1002/mds.24064
PMCID: PMC3323119  PMID: 22287014
Cerebellar Ataxia; Spinocerebellar Ataxia; Dominant Genetic Conditions; Recessive Genetic Conditions; Copy Number Variation
24.  Cerebrospinal Fluid Biomarkers and Proximity to Diagnosis in Preclinical Familial Alzheimer's Disease 
Background/Aims:
Biological markers of utility in tracking Alzheimer's disease (AD) during the presymptomatic prodromal phase are important for prevention studies. Changes in cerebrospinal fluid (CSF) levels of 42-amino-acid β-amyloid (Aβ42), total tau protein (t-tau) and phosphorylated tau at residue 181 (p-tau181) during this state are incompletely characterized.
Methods
We measured CSF markers in 13 carriers of familial AD (FAD) mutations that are fully penetrant for causing AD (PSEN1 and APP) and in 5 non-mutation-carrying family members.
Results
Even among the entirely presymptomatic mutation carriers (n = 9), Aβ42 was diminished (388.7 vs. 618.4 pg/ml, p = 0.004), and t-tau (138.5 vs. 50.5 pg/ml, p = 0.002) and p-tau181 (71.7 vs. 24.6 pg/ml, p = 0.003) were elevated. There was a negative correlation between Aβ42 levels and age relative to the family-specific age of dementia diagnosis.
Conclusions
Our data are consistent with a decline in CSF Aβ42 levels occurring at least 20 years prior to clinical dementia in FAD.
doi:10.1159/000335729
PMCID: PMC3696356  PMID: 22343824
Cerebrospinal fluid; Biomarkers; Familial Alzheimer's disease, presymptomatic; PSEN1 gene; APP gene; tau protein; β-Amyloid, 42-amino-acid form
25.  Familial Cortical Myoclonus with a Mutation in NOL3 
Annals of neurology  2012;72(2):175-183.
Objective
Myoclonus is characterized by sudden, brief involuntary movements and its presence is debilitating. We identified a family suffering from adult-onset, cortical myoclonus without associated seizures. We performed clinical, electrophysiological, and genetic studies to define this phenotype.
Methods
A large, four-generation family with history of myoclonus underwent careful questioning, examination, and electrophysiological testing. Thirty-five family members donated blood samples for genetic analysis, which included SNP mapping, microsatellite linkage, targeted massively parallel sequencing, and Sanger sequencing. In silico and in vitro experiments were performed to investigate functional significance of the mutation.
Results
We identified 11 members of a Canadian Mennonite family suffering from adult-onset, slowly progressive, disabling, multifocal myoclonus. Somatosensory evoked potentials indicated a cortical origin of the myoclonus. There were no associated seizures. Some severely affected individuals developed signs of progressive cerebellar ataxia of variable severity late in the course of their illness. The phenotype was inherited in an autosomal dominant fashion. We demonstrated linkage to chromosome 16q21-22.1. We then sequenced all coding sequence in the critical region, identifying only a single co-segregating, novel, nonsynonymous mutation, which resides in the gene NOL3. Furthermore, this mutation was found to alter post-translational modification of NOL3 protein in vitro.
Interpretation
We propose that Familial Cortical Myoclonus (FCM) is a novel movement disorder that may be caused by mutation in NOL3. Further investigation of the role of NOL3 in neuronal physiology may shed light on neuronal membrane hyperexcitability and pathophysiology of myoclonus and related disorders.
doi:10.1002/ana.23666
PMCID: PMC3431191  PMID: 22926851

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