Comparison of an expanded ataxia interactome with patient medical records reveals a relationship between macular degeneration and ataxia

Spinocerebellar ataxias 6 and 7 (SCA6 and SCA7) are neurodegenerative disorders caused by expansion of CAG repeats encoding polyglutamine (polyQ) tracts in CACNA1A, the alpha1A subunit of the P/Q-type calcium channel, and ataxin-7 (ATXN7), a component of a chromatin-remodeling complex, respectively. We hypothesized that finding new protein partners for ATXN7 and CACNA1A would provide insight into the biology of their respective diseases and their relationship to other ataxia-causing proteins. We identified 118 protein interactions for CACNA1A and ATXN7 linking them to other ataxia-causing proteins and the ataxia network. To begin to understand the biological relevance of these protein interactions within the ataxia network, we used OMIM to identify diseases associated with the expanded ataxia network. We then used Medicare patient records to determine if any of these diseases co-occur with hereditary ataxia. We found that patients with ataxia are at 3.03-fold greater risk of these diseases than Medicare patients overall. One of the diseases comorbid with ataxia is macular degeneration (MD). The ataxia network is significantly (P= 7.37 × 10−5) enriched for proteins that interact with known MD-causing proteins, forming a MD subnetwork. We found that at least two of the proteins in the MD subnetwork have altered expression in the retina of Ataxin-7266Q/+ mice suggesting an in vivo functional relationship with ATXN7. Together these data reveal novel protein interactions and suggest potential pathways that can contribute to the pathophysiology of ataxia, MD, and diseases comorbid with ataxia

Ataxin1L Is a Regulator of HSC Function Highlighting the Utility of Cross-Tissue Comparisons for Gene Discovery

<div><p>Hematopoietic stem cells (HSCs) are rare quiescent cells that continuously replenish the cellular components of the peripheral blood. Observing that the ataxia-associated gene <i>Ataxin-1-like</i> (<i>Atxn1L</i>) was highly expressed in HSCs, we examined its role in HSC function through <i>in vitro</i> and <i>in vivo</i> assays. Mice lacking Atxn1L had greater numbers of HSCs that regenerated the blood more quickly than their wild-type counterparts. Molecular analyses indicated <i>Atxn1L</i> null HSCs had gene expression changes that regulate a program consistent with their higher level of proliferation, suggesting that <i>Atxn1L</i> is a novel regulator of HSC quiescence. To determine if additional brain-associated genes were candidates for hematologic regulation, we examined genes encoding proteins from autism- and ataxia-associated protein–protein interaction networks for their representation in hematopoietic cell populations. The interactomes were found to be highly enriched for proteins encoded by genes specifically expressed in HSCs relative to their differentiated progeny. Our data suggest a heretofore unappreciated similarity between regulatory modules in the brain and HSCs, offering a new strategy for novel gene discovery in both systems.</p> </div

Loss of <i>Atxn1L</i> results in more proliferative hematopoietic stem and progenitor cells.

<p>A. Individual HSCs were sorted into 96-well plates containing methylcellulose media and colonies were counted and scored based on their morphology at the indicated time points. B. Proportions of colony types. C. Colony numbers from BM cells transduced with <i>Atxn1L</i>-overexpressing retrovirus compared to a GFP-only control vector (E.V). Results represent the average of three 96-well plates (<i>P<0.01</i>). D and E. <i>In vivo</i> proliferation analysis of WT vs <i>Atxn1L^−/−</i> HSCs (D) (KSL, Flk2⁻, CD34⁻) and hematopoietic progenitors (E) (KSL) by Ki67 staining (n = 5, <i>P<0.05</i>). F. Representative flow cytometry plots of Ki67 staining on hematopoietic progenitors (KSL). G. Complete blood counts over time of blood from WT and <i>Atxn1L^−/−</i> mice after a single injection of 5-FU. (n = 10/genotype, <i>P = 0.0007</i>). The graph shows representative data from three independent experiments. All graphs display the mean plus standard error.</p

<i>Atxn1L^−/−</i> mice have enhanced HSC function.

<p>A. Schematic of the experimental design. Equal numbers of bone marrow (BM) cells from WT (competitors) and <i>Atxn1L</i>^−/− or WT (donor) cells were transplanted into lethally irradiated recipients. For secondary transplants, HSCs were purified from primary recipients and transplanted into new recipients along with fresh whole BM competitor cells. B. Competitive whole BM transplants comparing the engraftment ability of WT vs <i>Atxn1L^−/−</i> cells. White and grey bars indicate peripheral blood contribution at 4 and 16 weeks post transplant. Red bars indicate donor cell contribution to bone marrow after 16 weeks. C. Peripheral blood chimerism after purified HSC transplantation at the indicated weeks. Twenty purified HSCs from WT and <i>Atxn1L^−/−</i>mice were transplanted along with 250,000 WT competitor BM cells. D. Analysis of the proportion of donor-derived HSCs obtained from transplant recipients as pooled from 5 mice. HSCs were defined as SP^KSL+CD150+ cells. E. Peripheral blood chimerism after secondary transplants from HSC-transplanted mice from (D) at the indicated weeks (n = 5). F. Limiting dilution competitive repopulation assay with the indicated numbers of WT and <i>Atxn1L^−/−</i> BM cells. The table shows the number of mice tested in each group and the number of mice that were engrafted with donor cells (contribution to blood>0.1%). G. The graph shows the percentage of mice that contain less than 0.1% multi-lineage engraftment 12 weeks post transplant. The HSC frequency was calculated using the L-Calc software according to Poisson statistics (two-tailed t-test; p = 0.016). (* <i>P<0.05</i>, ** <i>P<0.01</i>). All bone marrow transplantation experiments were repeated at least twice with similar results. All graphs display the mean plus standard error.</p

An unexpected relationship between the hematopoietic and nervous systems.

<p>A. Enrichment scores (a standardized measure of deviation between the observed overlap and expected overlap of two sets: ((observed – expected)/StDev(observed)), see methods for details) for the indicated gene sets compared to genes reported to have behavioral/neurological or nervous system phenotypes in the Mouse Genome Informatics (MGI) database; Fisher's exact tests were used to determine p-values. B. Analysis of the correlation between reported phenotypes across all genes from the MGI database, with −1 (blue) representing negative correlation, and 1 (yellow) representing high correlation. C. Enrichment analysis (same as in A above) for genes expressed in HSCs compared to the indicated gene sets. D. HSC expressed genes were mapped to the ataxia interactome. This subnetwork consists of HSC expressed nodes and their connected interacting partners that form the ataxia interactome. HSC expressed proteins are pink, HSC fingerprint proteins are red and black nodes are interacting partners. The node size corresponds to its connectedness with the rest of the network.</p

Expression of <i>Atxn1L</i> in the hematopoietic system and frequency of progenitors in knock-out animals.

<p>A. Real-time rt-PCR analysis of <i>Atxn1L</i> expression in the indicated purified populations, normalized to the level in MPPs. LT-HSC: long-term hematopoietic stem cells; MPP: multipotent progenitors (MPP). Terminally differentiated cells were purified from the peripheral blood. B. Real-time rt-PCR analysis of expression of the indicated genes in purified LT-HSCs, normalized to <i>Scl/Tal1</i>. Data are representative of two independent experiments. C, D. Analysis of the proportion of the indicated populations in the bone marrow of WT vs <i>Atxn1L^−/−</i> mice. CLP: common lymphoid progenitors, CMP: common myeloid progenitors, GMP: common myeloid progenitors, MEP: megakaryocyte-erythroid progenitors. E. Analysis of the proportion of LT-HSCs cells in the bone marrow of WT vs. <i>Atxn1L^−/−</i> mice (t-test, <i>P = 0.047</i>). F, Analysis of the proportion of ST-HSCs and MPPs. ST-HSC: short-term HSCs. LT-HSCs: Lineage⁻, Sca-1⁺, c-kit⁺, CD34⁻, Flt3⁻; other populations defined as in methods. In C–F, n≥5; bars indicate the mean plus standard error.</p

<i>Atxn1L^−/−</i> HSCs are enriched for expression of HSC-specific genes and depleted for quiescence-associated genes.

<p>A. Heat map showing the expression profiles for each biological replicate from the microarray. B. Count of the expected and observed number of genes overlapping between the published HSC fingerprint genes <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003359#pgen.1003359-Chambers1" target="_blank">[7]</a> and genes that are differentially expressed between WT and <i>Atxn1L^−/−</i> HSCs. (<i>P = 0.004152</i>) C. Count of the expected and observed number of genes overlapping between the published Quiescence-signature genes and genes that are down-regulated in <i>Atxn1L^−/−</i> HSCs compared to WT (**, <i>P = 2.044×10⁻⁵</i>) and of the genes overlapping between the published Proliferation-signature genes and those that are up-regulated in <i>Atxn1L^−/−</i> HSCs compared to WT. (*, <i>P = 0.06645</i>). HSCs were purified from 8-week-old mice and pooled for each chip.</p

Genes present in the autism interactome, the ataxia interactome, and the HSC fingerprint.

<p>Unk: Unknown.</p

Loss of <i>Atxn1L</i> does not affect the ability of HSCs to home to the recipient marrow after transplantation.

<p>A. Schematic of experimental design. 30,000 CD45.2 hematopoietic progenitors (lin−, Sca-1+, c-kit+, [KSL]) were purified and transplanted into CD45.1 recipient mice (n = 7 per genotype). (B) 18 hours post transplant, bone marrow from the recipient mice was analyzed by flow cytometry for the presence of CD45.2 donor-derived cells. Representative of two independent experiments. C. The graph shows the absolute number of WT vs Atxn1L^−/− donor-derived cells in the BM of recipient mice. (Not significantly different.) D. A portion of the KSL cells that were transplanted into CD45.1 recipient mice was also plated on methylcellulose medium to assess cell proliferation. The graph shows the average number of colonies from triplicate plates derived from WT vs. Atxn1L^−/− progenitor cells after 12 days in culture (<i>P<0.01</i>). Error bars indicate standard error of the mean.</p