Mapping of the spontaneous deletion in the Ap3d1 gene of mocha mice: fast and reliable genotyping

<p>Abstract</p> <p>Background</p> <p>The <it>mocha </it>mouse carries a spontaneous deletion in the <it>Ap3d1 </it>gene, encoding the delta 1 subunit of the adaptor related protein complex 3, (Ap3d1), and subsequently lack the expression of functional AP-3. This leads to a deficiency in vesicle transport and storage, which affects neurotransmitter vesicle turnover and release in the central nervous system. Since the genomic sequence of the <it>Ap3d1 </it>gene of <it>mocha </it>mouse is not known, precise mapping of the deletion as well as reliable genotyping protocols are lacking.</p> <p>Findings</p> <p>We sequenced the <it>Ap3d1 </it>gene (HGNC GeneID: 8943) around the deletion site in the <it>mocha </it>mouse and revealed a 10639 bp deletion covering exon 2 to 6. Subsequently, new PCR primers were designed yielding a reliable genotyping protocol of both newborn and adult tissue. To examine the genotypes further, hippocampal neurons were cultured from <it>mocha </it>and control mice. Patch-clamp recordings showed that <it>mocha </it>neurons had a higher input resistance, and that autaptic EPSC in <it>mocha </it>cultures depressed faster and stronger as compared with control cultures.</p> <p>Conclusion</p> <p>Our study reports the sequence of the deleted part of the <it>Ap3d1 </it>gene in <it>mocha </it>mice, as well as a reliable PCR-based genotyping protocol. We cultured hippocampal neurons from control and <it>mocha </it>mice, and found a difference in input resistance of the neurons, and in the synaptic short-term plasticity of glutamatergic autapses showing a larger synaptic depression than controls. The described procedures may be useful for the future utilization of the <it>mocha </it>mouse as a model of defective vesicle biogenesis. Importantly, as genotyping by eye color is complicated in newborn mice, the designed protocol is so fast and reliable that newborn mice could rapidly be genotyped and hippocampal neurons dissociated and cultured, which is normally best done at P0-P2.</p

Neonatal AAV delivery of alpha-synuclein induces pathology in the adult mouse brain

Abstract Abnormal accumulation of alpha-synuclein (αsyn) is a pathological hallmark of Lewy body related disorders such as Parkinson’s disease and Dementia with Lewy body disease. During the past two decades, a myriad of animal models have been developed to mimic pathological features of synucleinopathies by over-expressing human αsyn. Although different strategies have been used, most models have little or no reliable and predictive phenotype. Novel animal models are a valuable tool for understanding neuronal pathology and to facilitate development of new therapeutics for these diseases. Here, we report the development and characterization of a novel model in which mice rapidly express wild-type αsyn via somatic brain transgenesis mediated by adeno-associated virus (AAV). At 1, 3, and 6 months of age following intracerebroventricular (ICV) injection, mice were subjected to a battery of behavioral tests followed by pathological analyses of the brains. Remarkably, significant levels of αsyn expression are detected throughout the brain as early as 1 month old, including olfactory bulb, hippocampus, thalamic regions and midbrain. Immunostaining with a phospho-αsyn (pS129) specific antibody reveals abundant pS129 expression in specific regions. Also, pathologic αsyn is detected using the disease specific antibody 5G4. However, this model did not recapitulate behavioral phenotypes characteristic of rodent models of synucleinopathies. In fact no deficits in motor function or cognition were observed at 3 or 6 months of age. Taken together, these findings show that transduction of neonatal mouse with AAV-αsyn can successfully lead to rapid, whole brain transduction of wild-type human αsyn, but increased levels of wildtype αsyn do not induce behavior changes at an early time point (6 months), despite pathological changes in several neurons populations as early as 1 month

Additional file 1: Figure S1. of Neonatal AAV delivery of alpha-synuclein induces pathology in the adult mouse brain

Representative intensity of Human αsyn immunostaining (a) Photomicrographs representative of the variability of expression observed in the different group of animals at 1, 3 and 6 months of age (b) Level of expression of the transgene was assessed by western blot in AAV-αsyn at 3 months of age and compared to transgenic mice overexpressing αsyn under Thy1 promoter (line 61) at the same age. Antibody recognizing human and mouse αsyn was used (clone 42). (c) Quantification of the western blot shows αsyn level increase of 2.93 ± 0.33 fold in the AAV-αsyn animals and 3.23 ± 0.12 fold in the line 61. .The data are expressed as the amount of total level of αsyn normalized to actin (*, p < 0.05) and are from 3 repeated experiments. (PDF 1678 kb

Additional file 2: Figure S2. of Neonatal AAV delivery of alpha-synuclein induces pathology in the adult mouse brain

Neither ThioS positive structures nor neurodegeneration are observed in AAV-αsyn animals. (a-i) Sagittal brain sections were incubated with anti human asyn antibody followed by 5 min in 1% thioS solution. Thalamus (a-c) and cortex (d-f) of AAV-asyn animal show strong asyn immunoreactivity (a, d) that is not thioS- positive (b, e). As a control, human DLBD brain was co-stained in parallel. Cortical Lewy bodies positive for human asyn (g) are reactive to thioS (h, i). Representative images of NeuN-labeled cells in the cortex of AAV-asyn (n = 9) and AAV-venus (n = 7) at 6 months of age (k). Quantification of NeuN-positive cells in the whole cortex (area delineated in blue). Data are presented as as mean ± S.E.M means. Scale bars in i = 40 μm and applied to a-h; Scale bars in k = 2 mm. Abbreviation: DLBD; Diffuse Lewy Body Disease. (PDF 1541 kb

Additional file 1 of A mutant methionyl-tRNA synthetase-based toolkit to assess induced-mesenchymal stromal cell secretome in mixed-culture disease models

Additional file 1: Fig. S1. Uncropped agarose gel from Figure 1B