Altered Dendritic Morphology of Purkinje cells in Dyt1 ΔGAG Knock-In and Purkinje Cell-Specific Dyt1 Conditional Knockout Mice

BACKGROUND: DYT1 early-onset generalized dystonia is a neurological movement disorder characterized by involuntary muscle contractions. It is caused by a trinucleotide deletion of a GAG (ΔGAG) in the DYT1 (TOR1A) gene encoding torsinA; the mouse homolog of this gene is Dyt1 (Tor1a). Although structural and functional alterations in the cerebellum have been reported in DYT1 dystonia, neuronal morphology has not been examined in vivo. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we examined the morphology of the cerebellum in Dyt1 ΔGAG knock-in (KI) mice. Golgi staining of the cerebellum revealed a reduction in the length of primary dendrites and a decrease in the number of spines on the distal dendrites of Purkinje cells. To determine if this phenomenon was cell autonomous and mediated by a loss of torsinA function in Purkinje cells, we created a knockout of the Dyt1 gene only in Purkinje cells of mice. We found the Purkinje-cell specific Dyt1 conditional knockout (Dyt1 pKO) mice have similar alterations in Purkinje cell morphology, with shortened primary dendrites and decreased spines on the distal dendrites. CONCLUSION/SIGNIFICANCE: These results suggest that the torsinA is important for the proper development of the cerebellum and a loss of this function in the Purkinje cells results in an alteration in dendritic structure

Enhanced Hippocampal Long-Term Potentiation and Fear Memory in Btbd9 Mutant Mice

Polymorphisms in BTBD9 have recently been associated with higher risk of restless legs syndrome (RLS), a neurological disorder characterized by uncomfortable sensations in the legs at rest that are relieved by movement. The BTBD9 protein contains a BTB/POZ domain and a BACK domain, but its function is unknown. To elucidate its function and potential role in the pathophysiology of RLS, we generated a line of mutant Btbd9 mice derived from a commercial gene-trap embryonic stem cell clone. Btbd9 is the mouse homolog of the human BTBD9. Proteins that contain a BTB/POZ domain have been reported to be associated with synaptic transmission and plasticity. We found that Btbd9 is naturally expressed in the hippocampus of our mutant mice, a region critical for learning and memory. As electrophysiological characteristics of CA3-CA1 synapses of the hippocampus are well characterized, we performed electrophysiological recordings in this region. The mutant mice showed normal input-output relationship, a significant impairment in pre-synaptic activity, and an enhanced long-term potentiation. We further performed an analysis of fear memory and found the mutant mice had an enhanced cued and contextual fear memory. To elucidate a possible molecular basis for these enhancements, we analyzed proteins that have been associated with synaptic plasticity. We found an elevated level of dynamin 1, an enzyme associated with endocytosis, in the mutant mice. These results suggest the first identified function of Btbd9 as being involved in regulating synaptic plasticity and memory. Recent studies have suggested that enhanced synaptic plasticity, analogous to what we have observed, in other regions of the brain could enhance sensory perception similar to what is seen in RLS patients. Further analyses of the mutant mice will help shine light on the function of BTBD9 and its role in RLS

Alternating Hemiplegia of Childhood-Related Neural and Behavioural Phenotypes in Na+,K+-ATPase α3 Missense Mutant Mice

Missense mutations in ATP1A3 encoding Na(+),K(+)-ATPase α3 have been identified as the primary cause of alternating hemiplegia of childhood (AHC), a motor disorder with onset typically before the age of 6 months. Affected children tend to be of short stature and can also have epilepsy, ataxia and learning disability. The Na(+),K(+)-ATPase has a well-known role in maintaining electrochemical gradients across cell membranes, but our understanding of how the mutations cause AHC is limited. Myshkin mutant mice carry an amino acid change (I810N) that affects the same position in Na(+),K(+)-ATPase α3 as I810S found in AHC. Using molecular modelling, we show that the Myshkin and AHC mutations display similarly severe structural impacts on Na(+),K(+)-ATPase α3, including upon the K(+) pore and predicted K(+) binding sites. Behavioural analysis of Myshkin mice revealed phenotypic abnormalities similar to symptoms of AHC, including motor dysfunction and cognitive impairment. 2-DG imaging of Myshkin mice identified compromised thalamocortical functioning that includes a deficit in frontal cortex functioning (hypofrontality), directly mirroring that reported in AHC, along with reduced thalamocortical functional connectivity. Our results thus provide validation for missense mutations in Na(+),K(+)-ATPase α3 as a cause of AHC, and highlight Myshkin mice as a starting point for the exploration of disease mechanisms and novel treatments in AHC

Genome-wide association study identifies multiple loci associated with both mammographic density and breast cancer risk

Mammographic density reflects the amount of stromal and epithelial tissues in relation to adipose tissue in the breast and is a strong risk factor for breast cancer. Here we report the results from meta-analysis of genome-wide association studies (GWAS) of three mammographic density phenotypes: dense area, non-dense area and percent density in up to 7,916 women in stage 1 and an additional 10,379 women in stage 2. We identify genome-wide significant (P<5×10−8) loci for dense area (AREG, ESR1, ZNF365, LSP1/TNNT3, IGF1, TMEM184B, SGSM3/MKL1), non-dense area (8p11.23) and percent density (PRDM6, 8p11.23, TMEM184B). Four of these regions are known breast cancer susceptibility loci, and four additional regions were found to be associated with breast cancer (P<0.05) in a large meta-analysis. These results provide further evidence of a shared genetic basis between mammographic density and breast cancer and illustrate the power of studying intermediate quantitative phenotypes to identify putative disease susceptibility loci

Purkinje cells in KI mice.

<p>(A) A representative Purkinje cell trace (left). A representative Purkinje cell from CT and KI mice at 40× magnification (right). (B) There was no significant difference in size of the Purkinje cell soma between CT and KI mice. (C) However, the large primary dendrite of the Purkinje cells in the KI mice was significantly shorter than those in CT mice. (D) Next, the quaternary dendrite branch of CT and KI mice was examined at 100× magnification. (E) The number of spines on the quaternary dendrite branch in the KI mice was significantly reduced compared to CT mice. Scale bars in Panel A represent 10 µm. Scale bars in panel D represent 1 µm. Bars in Panels B, C and E represent means with standard errors. ** p<0.01.</p

Purkinje cells in <i>Dyt1</i> pKO mice.

<p>(A) A representative Purkinje cell from control and <i>Dyt1</i> pKO mice, produced at 40× magnification. (B) The size of the Purkinje cell soma in <i>Dyt1</i> pKO mice was not significantly different than those of CT mice. (C) The large primary dendrite of the Purkinje cells in the <i>Dyt1</i> pKO mice was significantly shorter than those in CT mice. (D) A representative quaternary dendrite branch of CT and <i>Dyt1</i> pKO mice was examined at 100× magnification. (E) The number of spines on the quaternary dendrite branch in the <i>Dyt1</i> pKO mice were significantly reduced compared to those of CT mice. Scale bars in Panel A represent 10 µm. Scale bars in Panel D represent 1 µm. Bars in Panels B, C and E represent means with standard errors. ** p<0.01, *** p<0.001.</p

Generation of <i>Dyt1</i> pKO mice.

<p>(A) Schematic diagram of the generation of the <i>Dyt1</i> pKO mice. Filled boxes represent exons. Filled triangles indicate <i>loxP</i> sites. Open triangles indicate the <i>FRT</i> sites that were incorporated to remove the <i>neo</i> cassette. <i>Dyt1 loxP</i> mice were crossed with <i>Pcp2-cre</i> mice to obtain double heterozygotes. The double heterozygotes were crossed with <i>Dyt1 loxP</i> homozygotes to obtain <i>Dyt1</i> pKO mice. The primer sites for genotyping of <i>Dyt1</i> locus were shown by an arrow pairs. The short bar under exon 5 is the site of probe used for <i>in situ</i> hybridization. <i>Dyt1</i> exons 3 and 4 were removed in Purkinje cells of <i>Dyt1</i> pKO mice. (B) An agarose gel showing the various PCR products that were used to genotype mice. The top band indicates the presence of the <i>Pcp2-cre</i> locus, the middle band represents the <i>Dyt1 loxP</i> locus, and the bottom band represents the <i>Dyt1</i> wild-type locus. Lanes 4: <i>Dyt1 loxP</i> homozygous mice. Lanes 2, 6: <i>Dyt1 loxP</i> heterozygous mice. Lanes 1, 3, 5: <i>Dyt1</i> pKO mice. (C) <i>In situ</i> hybridization was used to confirm the Purkinje cell-specific knockout of the <i>Dyt1</i> gene. CT (C.1) and <i>Dyt1</i> pKO (C.2) mice. GL: granule cell layer; PC: Purkinje cell layer; ML: molecular layer. Scale bar represents 100 µm in C.2.</p

Freezing behavior in fear conditioning experiment.

<p>(<b>A</b>) <i>Btbd9</i> mutant mice and WT mice were conditioned to two 30-second tones followed by an electric shock, with a shock interval of 120 seconds. Solid black rectangle represents the period of tone. The vertical line at represents the period of the shock. (<b>B</b>) <i>Btbd9</i> mutant mice had increased percentage of freezing behavior in both contextual and cued fear conditioning, suggesting that the <i>Btbd9</i> mutant mice had enhanced fear memory. Vertical bars represent means ± SEM. * p<0.05.</p

Hippocampal CA1 electrophysiological field recordings.

<p>(<b>A</b>) <i>Btbd9</i> mutant mice showed no differences in input-output relationships. Small inset graph is a representative trace with varying stimulus intensities. (<b>B</b>) <i>Btbd9</i> mutant mice showed enhanced paired-pulse ratios at three inter-stimuli values. Panel above graph are representative traces at the three inter-stimuli values that were significantly different between <i>Btbd9</i> mutant and WT mice. (<b>C</b>) <i>Btbd9</i> mutant mice showed enhanced late long-term potentiation. Small inset graphs are representative traces, with red signifying before LTP induction and black signifying after LTP induction. Circles represent means ± SEM. * p<0.05.</p