Multiparametric graph theoretical analysis reveals altered structural and functional network topology in Alzheimer's disease

Alzheimer's disease (AD), an irreversible neurodegenerative disease, is the most common type of dementia in elderly people. This present study incorporated multiple structural and functional connectivity metrics into a graph theoretical analysis framework and investigated alterations in brain network topology in patients with mild cognitive impairment (MCI) and AD. By using this multiparametric analysis, we expected different connectivity metrics may reflect additional or complementary information regarding the topological changes in brain networks in MCI or AD. In our study, a total of 73 subjects participated in this study and underwent the magnetic resonance imaging scans. For the structural network, we compared commonly used connectivity metrics, including fractional anisotropy and normalized streamline count, with multiple diffusivity-based metrics. We compared Pearson correlation and covariance by investigating their sensitivities to functional network topology. Significant disruption of structural network topology in MCI and AD was found predominantly in regions within the limbic system, prefrontal and occipital regions, in addition to widespread alterations of local efficiency. At a global scale, our results showed that the disruption of the structural network was consistent across different edge definitions and global network metrics from the MCI to AD stages. Significant changes in connectivity and tract-specific diffusivity were also found in several limbic connections. Our findings suggest that tract-specific metrics (e.g., fractional anisotropy and diffusivity) provide more sensitive and interpretable measurements than does metrics based on streamline count. Besides, the use of inversed radial diffusivity provided additional information for understanding alterations in network topology caused by AD progression and its possible origins. Use of this proposed multiparametric network analysis framework may facilitate early MCI diagnosis and AD prevention. Keywords: Alzheimer's disease, Mild cognitive impairment, Diffusion tensor imaging, Resting-state functional MRI, Brain network, Structural connectivity, Functional connectivity, Graph theoretical analysi

Effects on Murine Behavior and Lifespan of Selectively Decreasing Expression of Mutant Huntingtin Allele by Supt4h Knockdown

<div><p>Production of protein containing lengthy stretches of polyglutamine encoded by multiple repeats of the trinucleotide CAG is a hallmark of Huntington’s disease (HD) and of a variety of other inherited degenerative neurological and neuromuscular disorders. Earlier work has shown that interference with production of the transcription elongation protein SUPT4H results in decreased cellular capacity to transcribe mutant huntingtin gene (<i>Htt</i>) alleles containing long CAG expansions, but has little effect on expression of genes containing short CAG stretches. zQ175 and R6/2 are genetically engineered mouse strains whose genomes contain human <i>HTT</i> alleles that include greatly expanded CAG repeats and which are used as animal models for HD. Here we show that reduction of SUPT4H expression in brains of zQ175 mice by intracerebroventricular bolus injection of antisense 2’-<i>O</i>-methoxyethyl oligonucleotides (ASOs) directed against <i>Supt4h</i>, or in R6/2 mice by deletion of one copy of the <i>Supt4h</i> gene, results in a decrease in mRNA and protein encoded specifically by mutant <i>Htt</i> alleles. We further show that reduction of SUPT4H in mouse brains is associated with decreased HTT protein aggregation, and in R6/2 mice, also with prolonged lifespan and delay of the motor impairment that normally develops in these animals. Our findings support the view that targeting of SUPT4H function may be useful as a therapeutic countermeasure against HD.</p></div

Mutant HTT aggregation in the brain of R6/2 mice deleted for one <i>Supt4h</i> allele.

<p>(A) Wild-type HTT protein levels were examined by Western blot analysis using brain lysates collected from right cerebrum of animals as described in Materials and Methods and in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005043#pgen.1005043.g003" target="_blank">Fig 3B</a>. TATA-binding protein (TBP) produced by a gene containing 13–15 consecutive CAA/CAG repeats was also analyzed. β-actin served as loading control. (B) Brain lysates collected from 12-week-old mice were loaded onto a cellulose acetate CA membrane, which traps only aggregated protein. Mutant HTT protein was detected using EM48 antibody. Nitrocellulose (NC) membranes were employed for slot blot assays to determine protein abundance; α-Tubulin served as a loading control. The values shown are means ± SEM, and the relative protein aggregation in tissues of R6/2 HD mice having two or one allele of <i>Supt4h</i> is presented in the bottom panel. (C) Representative IHC images of cerebral tissue of 12-week-old R6/2 (HD) mice having either one or two alleles of <i>Supt4h</i> are shown. HTT aggregates were detected using an antibody against ubiquitin, which is recruited to and co-localized with aggregates in the brain of HD mice [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005043#pgen.1005043.ref054" target="_blank">54</a>]. The positions of aggregates are indicated by arrowheads. (D) DARPP-32 protein abundance was analyzed by Western blot analysis using brain lysates collected from R6/2 mice at the age of 12 weeks either intact in the <i>Supt4h</i> locus or deleted for one <i>Supt4h</i> allele. The level of WT mice having two <i>Supt4h</i> alleles was set to 1, after normalization with α-Tubulin. Data are presented as the mean ± SEM (n = 3 in each group; *, <i>p</i> < 0.05 by Student’s <i>t</i>-test).</p

Effect of <i>Supt4h</i> deletion on motor function and lifespan of R6/2 mice.

<p>(A) Mice at the age of 8 to 14 weeks were tested for rotarod performance as described in Materials and Methods. (B) Latency of indicated animals, at the age of 6 to 14 weeks, on beam walking was analyzed. Data are presented as the mean ± SEM. *, <i>p</i> < 0.05; ***, <i>p</i> < 0.001 in comparison with R6/2 <i>S</i>^+/+ mice, using two-way ANOVA followed by Bonferroni <i>post hoc</i> test. (C) Longevity of indicated animals was recorded. Compared to R6/2 <i>S</i>^+/+ mice, HD animals with a single allele of <i>Supt4h</i> deletion (R6/2 <i>S</i>^+/-) showed a longer lifespan (<i>p</i> = 0.0204, Log-rank test). (D) Body weight was measured weekly and its change relative to the body weight at the age of 7 weeks is shown. Results were collected from mice at 7 through 15 weeks of age. The number of mice (n) used in each individual assay is indicated.</p

Creation and characterization of <i>Supt4h</i> knockout mice.

<p>(A) Genomic organization of the mouse <i>Supt4h</i> locus (Top) and structure of the targeting vector (Middle). In the allele carrying the <i>Supt4h</i> deletion, a neo cassette specifying resistance to the antibiotic G418 in animal cells replaced the DNA fragment encompassing exon 2 to exon 5 of <i>Supt4h</i> via homologous recombination (Bottom). Positions of 5’ and 3’ flanking probes used in Southern blot analysis, and predicted sizes of restriction fragments detected by these probes are shown. Genomic DNA of C57BL6/129 mice (<i>S</i>^<i>+/+</i>) and their <i>Supt4h</i>^<i>+/-</i> (<i>S</i>^<i>+/-</i>) littermates was subjected to Southern blot analysis using the 5’ and 3’ probes separately. (B) <i>Supt4h</i> mRNA levels were assessed by qRT-PCR using the brain tissue of <i>Supt4h</i>^<i>+/+</i> and <i>Supt4h</i>^<i>+/-</i> mice. The abundance in <i>Supt4h</i>^<i>+/+</i> mice was set as 1, after normalization with <i>U6</i> RNA. (C) SUPT4H protein level in the striatum and cortex of indicated mice was analyzed by immunohistochemistry (IHC) using antibody against SUPT4H. (D) Protein lysates collected from the cerebrum of indicated mice were analyzed by Western blot using anti-SUPT4H antibody. GAPDH served as loading control. Data are presented as the mean ± SEM (n = 3 in each group; *, <i>p</i> < 0.05; ***, <i>p</i> <0.001 by Student’s <i>t</i>-test). The mice were sacrificed at the age of 12 weeks for analyses.</p

Effect of down regulation by Supt4h ASO on expression of mutant and wild-type <i>Htt</i> alleles in zQ175 HD mice.

<p>Supt4h ASO was delivered to the brain of zQ175 HD mice by intracerebroventricular (ICV) bolus injection. ASO became distributed throughout CNS via cerebral spinal fluid circulation, and as observed previously [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005043#pgen.1005043.ref018" target="_blank">18</a>] the spinal cord most susceptible to its effects. Mice were sacrificed 4 weeks after a single injection at the age of 5.5 months and spinal cords were collected for analyses of ASO effects. (A) <i>Supt4h</i> transcript abundance was assessed by quantitative RT-PCR. mRNA level in tissue obtained from PBS-treated zQ175 mice (mock) was set to 1, and relative <i>Supt4h</i> mRNA level in tissue from ASO-treated animals is shown. (B) SUPT4H protein level in tissue analyzed in (A) for mRNA abundance was examined by Western blot analysis. After normalization using α-Tubulin, the protein level was compared to mock control. (C) Left, wild-type (WT) and mutant (Mut) <i>Htt</i> gene expression were assessed by qRT-PCR in Supt4h ASO-treated samples and compared to that of mock samples. The level of WT <i>Htt</i> mRNA in mock samples was set as 1, and <i>Htt</i> transcripts produced from the co-existing Mut allele were approximately 40% of WT mRNA obtained from zQ175 KI mice. Right, production of wild type and mutant <i>Htt</i> mRNAs following intracerebroventricular bolus injection of an ASO [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005043#pgen.1005043.ref018" target="_blank">18</a>] that targets both the WT and Mut alleles of <i>Htt</i>. The conditions used for injection and analysis in these experiments were identical for those employed for the ASO targeting <i>Supt4h</i> (n = 3 in each group; *, <i>p</i> <0.05; **, <i>p</i> < 0.01; ***, <i>p</i> <0.001 by Student’s <i>t</i> test).</p

Effect of heterozygous deletion of <i>Supt4h</i> on expression of mutant and wild-type <i>Htt</i> alleles in R6/2 mice.

<p>(A) Outline of procedures used to generate heterozygous deletion of <i>Supt4h</i> in R6/2 HD mice by crossing with <i>Supt4h</i>^<i>+/-</i> mice, followed by summary of biochemical and phenotypic analyses of their offspring. (B) Expression of wild-type murine <i>Htt</i> gene was assessed by qRT-PCR using <i>U6</i> as an internal control. Samples were collected from left cerebrum of indicated animals at the age of 12 weeks, and the gene expression in WT mice containing two functional <i>Supt4h</i> alleles was set as 1. (C) Same as (B), except that expression of mutant <i>Htt</i> allele was analyzed and mutant <i>Htt</i> expression in R6/2 mice containing two functional <i>Supt4h</i> alleles was set as 1. Data are presented as the mean ± SEM (n = 3 in each group; **, <i>p</i> <0.01 by Student’s <i>t</i>-test).</p