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- Open Access
Complement is dispensable for neurodegeneration in Niemann-Pick disease type C
© Lopez et al.; licensee BioMed Central Ltd. 2012
- Received: 29 June 2012
- Accepted: 30 August 2012
- Published: 17 September 2012
The immune system has been implicated in neurodegeneration during development and disease. In various studies, the absence of complement (that is, C1q deficiency) impeded the elimination of apoptotic neurons, allowing survival. In the genetic lysosomal storage disease Niemann-Pick C (NPC), caused by loss of NPC1 function, the expression of complement system components, C1q especially, is elevated in degenerating brain regions of Npc1 -/- mice. Here we test whether complement is mediating neurodegeneration in NPC disease.
In normal mature mice, C1q mRNA was found in neurons, particularly cerebellar Purkinje neurons (PNs). In Npc1 -/- mice, C1q mRNA was additionally found in activated microglia, which accumulate during disease progression and PN loss. Interestingly, C1q was not enriched on or near degenerating neurons. Instead, C1q was concentrated in other brain regions, where it partially co-localized with a potential C1q inhibitor, chondroitin sulfate proteoglycan (CSPG). Genetic deletion of C1q, or of the downstream complement pathway component C3, did not significantly alter patterned neuron loss or disease progression. Deletion of other immune response factors, a Toll-like receptor, a matrix metalloprotease, or the apoptosis facilitator BIM, also failed to alter neuron loss.
We conclude that complement is not involved in the death and clearance of neurons in NPC disease. This study supports a view of neuroinflammation as a secondary response with non-causal relationship to neuron injury in the disease. This disease model may prove useful for understanding the conditions in which complement and immunity do contribute to neurodegeneration in other disorders.
- Lysosomal storage disease
- Purkinje neurons
- Extracellular matrix
Elevated immune and inflammatory factors are suspect in causing or promoting neurodegeneration in several neurological disorders. For the neurodegenerative lysosomal storage disease Niemann-Pick C (NPC), multiple independent gene profile studies analyzing Npc1 -/- mouse tissues and patient blood samples have identified immune response and inflammation pathway genes as the largest group whose expression is modified during disease progression . Although these genes can be used as indicators of disease severity, the relevance of these inflammatory mediators to the pathology remains unclear. Previously, we observed that deletion of the inflammatory chemokine Ccl3 gene did not have the beneficial effect on neurodegeneration in NPC-diseased mice that was evident for another lysosomal storage disorder, Sandhoff disease . In addition to chemokines, complement components, Toll-like receptors, proteases, and apoptotic facilitators are also found to be elevated in the brains of Npc1 -/- mice. Components in these pathways could play critical roles in the disease progression. Here, we focus primarily on defining the role of the innate immune complement component C1q in NPC disease, since in other neurodegenerative scenarios C1q has been proposed to mediate synapse removal and mark apoptotic neurons for lysis and clearance [3–5].
Expression and localization of C1q in mice with NPC disease
NPC-induced patterned neurodegeneration continues despite genetic inactivation of complement
Loss of other immune-related components, or an apoptosis facilitator, does not alter patterned neuron loss in NPC mice
Deficiency in other immune response-relevant pathways does not alter neurodegeneration in NPC disease
Npc1 -/- & ____
Change in PN loss
Change in microglia
Change in %CD68b
P = 0.49
P = 0.88
P = 0.95
P = 0.17
Purkinje neuron (PN) rescue
P = 0.0057
In this study, we show that C1q and other immune factors do not facilitate the elimination of dying neurons in the disease. This study agrees with a commonly employed mouse model of Parkinson disease where microglial C1q is reported to not affect nigrostriatal dopaminergic injury . The cell-autonomous and genetically controlled neuron survival in the NPC mouse model [2, 17] provides a tool and opportunity for uncovering alternate non-apoptotic mechanisms of neuron death and clearance that may also occur in many other neurodegenerative disorders.
Mice were managed in accordance with Stanford University’s Administrative Panel on Laboratory Animal Care. Npc1 +/- mice were derived as previously reported  and crossed with C1qa or C3 knockout mice obtained at Stanford University . Tlr7, Mmp12, and Bcl2l11(Bim) knockout mice were obtained from the Jackson Laboratory. Unc93b1 3d  mutant mice were obtained from the Mutant Mouse Regional Resource Centers. Genotyping was performed as detailed in the references listed above. To minimize background discrepancies, sibling offspring were used for comparisons. For example, mixed FVB/B6 C1qa +/- ; Npc1 +/- mice were mated to produce offspring with genotypes Npc1 -/- ; C1qa +/+ and Npc1 -/- ; C1qa -/- . Isolated brains were fixed whole overnight at 4°C in 4% paraformaldehyde in phosphate buffer saline (PBS).
Immunofluorescent and imaging procedures were performed as previously described . Primary antibody sources are as follows: rat anti-C1q (Abcam), rat anti-CD68 (AbdSerotec), rabbit and mouse anti-Calbindin-D28K (Sigma), and mouse anti-CSPG (Millipore). Stainings include Hoechst (Invitrogen) and NeuroTrace 435/455 fluorescent Nissl stain (Invitrogen). ImageJ and GraphPad Prism software were used for measurements and statistics. Unless stated otherwise, means and standard deviations are reported.
DIG-labeled antisense probes for in situ detection of C1qa were designed as previously described . A total of 200 ng/mL of purified DIG-labeled RNA probe was hybridized to 50 μm thick vibratome tissue sections that were processed in RNAse-free 5x SSC buffer. Hybridization was performed at 60°C for 16 h in hybridization solution: 50% formamide, 5x SSC, 0.1% Tween-20, 500 ug/mL tRNA, 500ug/mL salmon sperm DNA, 50 ug/mL Heparin salt, and 0.5% SDS. Washes were done at 60°C for 15 min using hybridization buffer followed by 5x SCC and 0.2x SSC buffers with 0.1% Tween-20 (Sigma). Afterwards, PBS with 2% BSA and 0.2% Triton X-100 (Sigma) was used for incubating anti-Digoxigenin-AP, Fab fragments (Roche) overnight at 4°C. The NBT/BCIP or HNPP Fast Red reaction was then performed as commercially directed (Roche). To mark CD68-positive cells microglia with DAB (Sigma), 50 μm vibratome sections were first treated with 0.6% hydrogen peroxide in methanol for 30 min followed by incubation of anti-CD68 antibody in PBS with 2% BSA and 0.2% Triton X-100. Diethylpyrocarbonate (DEPC; Sigma) was present in 0.01% vol/vol concentration throughout the CD68 immunohistochemical procedure. Unlike anti-CD68, anti-D28K can be used after in situ hybridization for immunofluorescent detection of PNs.
We thank Dr Ben Barres at Stanford University School of Medicine, who provided the C1q and C3 knockout mice, C1q probe templates, and helpful discussions. This work was supported by the Ara Parseghian Medical Research Foundation; Howard Hughes Medical Institute; National Institutes of Health [R01 NS073691 to MPS, GM07790 and GM007276 to MEL.
- Cluzeau CV, Watkins-Chow DE, Fu R, Borate B, Yanjanin N, Dail MK, Davidson CD, Walkley SU, Ory DS, Wassif CA, Pavan WJ, Porter FD: Microarray expression analysis and identification of serum biomarkers for Niemann-Pick disease, type C1. Hum Mol Genet 2012, 21:3632–3646.View ArticlePubMedPubMed CentralGoogle Scholar
- Lopez ME, Klein AD, Hong J, Dimbil UJ, Scott MP: Neuronal and epithelial cell rescue resolves chronic systemic inflammation in the lipid storage disorder Niemann-Pick C. Hum Mol Genet 2012, 21:2946–2960.View ArticlePubMedPubMed CentralGoogle Scholar
- Stevens B, Allen NJ, Vazquez LE, Howell GR, Christopherson KS, Nouri N, Micheva KD, Mehalow AK, Huberman AD, Stafford B, Sher A, Litke AM, Lambris JD, Smith SJ, John SW, Barres BA: The classical complement cascade mediates CNS synapse elimination. Cell 2007, 131:1164–1178.View ArticlePubMedGoogle Scholar
- Perry VH, O’Connor V: C1q: the perfect complement for a synaptic feast? Nat Rev Neurosci 2008, 9:807–811.View ArticlePubMedGoogle Scholar
- Paidassi H, Tacnet-Delorme P, Garlatti V, Darnault C, Ghebrehiwet B, Gaboriaud C, Arlaud GJ, Frachet P, Paidassi H, Tacnet-Delorme P, Garlatti V, Darnault C, Ghebrehiwet B, Gaboriaud C, Arlaud GJ, Frachet P: C1q binds phosphatidylserine and likely acts as a multiligand-bridging molecule in apoptotic cell recognition. J Immunol 2008, 180:2329–2338.View ArticlePubMedPubMed CentralGoogle Scholar
- Liao G, Wen Z, Irizarry K, Huang Y, Mitsouras K, Darmani M, Leon T, Shi L, Bi X: Abnormal gene expression in cerebellum of Npc1-/- mice during postnatal development. Brain Res 2010, 1325:128–140.View ArticlePubMedGoogle Scholar
- Distl R, Kuban R, Komkov V, Kallwellis K, von Deimling A, Ohm TG: Differential gene expression in BALB/c Npc1−/− and Npc1+/+ cerebellum. NCBI GEO DataSets 2007. accession GSE5944.Google Scholar
- Lobsiger CS, Boillee S, Cleveland DW: Toxicity from different SOD1 mutants dysregulates the complement system and the neuronal regenerative response in ALS motor neurons. Proc Natl Acad Sci U S A 2007, 104:7319–7326.View ArticlePubMedPubMed CentralGoogle Scholar
- Ko DC, Milenkovic L, Beier SM, Manuel H, Buchanan J, Scott MP: Cell-autonomous death of cerebellar purkinje neurons with autophagy in Niemann-Pick type C disease. PLoS Genet 2005, 1:81–95.View ArticlePubMedGoogle Scholar
- Bellander BM, Singhrao SK, Ohlsson M, Mattsson P, Svensson M: Complement activation in the human brain after traumatic head injury. J Neurotrauma 2001, 18:1295–1311.View ArticlePubMedGoogle Scholar
- Zhao M, Choi YS, Obrietan K, Dudek SM: Synaptic plasticity (and the lack thereof) in hippocampal CA2 neurons. J Neurosci 2007, 27:12025–12032.View ArticlePubMedGoogle Scholar
- Foscarin S, Ponchione D, Pajaj E, Leto K, Gawlak M, Wilczynski GM, Rossi F, Carulli D: Experience-dependent plasticity and modulation of growth regulatory molecules at central synapses. PLoS One 2011, 6:e16666.View ArticlePubMedPubMed CentralGoogle Scholar
- Kirschfink M, Blase L, Engelmann S, Schwartz-Albiez R: Secreted chondroitin sulfate proteoglycan of human B cell lines binds to the complement protein C1q and inhibits complex formation of C1. J Immunol 1997, 158:1324–1331.PubMedGoogle Scholar
- Ilieva H, Polymenidou M, Cleveland DW: Non-cell autonomous toxicity in neurodegenerative disorders: ALS and beyond. J Cell Biol 2009, 187:761–772.View ArticlePubMedPubMed CentralGoogle Scholar
- Sarna JR, Larouche M, Marzban H, Sillitoe RV, Rancourt DE, Hawkes R: Patterned Purkinje cell degeneration in mouse models of Niemann-Pick type C disease. J Comp Neurol 2003, 456:279–291.View ArticlePubMedGoogle Scholar
- Sillitoe RV, Vogel MW, Joyner AL: Engrailed homeobox genes regulate establishment of the cerebellar afferent circuit map. J Neurosci 2010, 30:10015–10024.View ArticlePubMedPubMed CentralGoogle Scholar
- Lopez ME, Klein AD, Dimbil UJ, Scott MP: Anatomically defined neuron-based rescue of neurodegenerative Niemann-Pick type C disorder. J Neurosci 2011, 31:4367–4378.View ArticlePubMedPubMed CentralGoogle Scholar
- Lund JM, Alexopoulou L, Sato A, Karow M, Adams NC, Gale NW, Iwasaki A, Flavell RA: Recognition of single-stranded RNA viruses by Toll-like receptor 7. Proc Natl Acad Sci U S A 2004, 101:5598–5603.View ArticlePubMedPubMed CentralGoogle Scholar
- Tabeta K, Hoebe K, Janssen EM, Du X, Georgel P, Crozat K, Mudd S, Mann N, Sovath S, Goode J, Shamel L, Herskovits AA, Portnoy DA, Cooke M, Tarantino LM, Wiltshire T, Steinberg BE, Grinstein S, Beutler B: The Unc93b1 mutation 3d disrupts exogenous antigen presentation and signaling via Toll-like receptors 3, 7 and 9. Nat Immunol 2006, 7:156–164.View ArticlePubMedGoogle Scholar
- Shipley JM, Wesselschmidt RL, Kobayashi DK, Ley TJ, Shapiro SD: Metalloelastase is required for macrophage-mediated proteolysis and matrix invasion in mice. Proc Natl Acad Sci U S A 1996, 93:3942–3946.View ArticlePubMedPubMed CentralGoogle Scholar
- Bouillet P, Metcalf D, Huang DC, Tarlinton DM, Kay TW, Kontgen F, Adams JM, Strasser A: Proapoptotic Bcl-2 relative Bim required for certain apoptotic responses, leukocyte homeostasis, and to preclude autoimmunity. Science 1999, 286:1735–1738.View ArticlePubMedGoogle Scholar
- Bouillet P, Robati M, Adams JM, Strasser A: Loss of pro-apoptotic BH3-only Bcl-2 family member Bim does not protect mutant Lurcher mice from neurodegeneration. J Neurosci Res 2003, 74:777–781.View ArticlePubMedGoogle Scholar
- Depboylu C, Schorlemmer K, Klietz M, Oertel WH, Weihe E, Hoglinger GU, Schafer MK: Upregulation of microglial C1q expression has no effects on nigrostriatal dopaminergic injury in the MPTP mouse model of Parkinson disease. J Neuroimmunol 2011, 236:39–46.View ArticlePubMedGoogle Scholar
- Botto M, Dell'Agnola C, Bygrave AE, Thompson EM, Cook HT, Petry F, Loos M, Pandolfi PP, Walport MJ: Homozygous C1q deficiency causes glomerulonephritis associated with multiple apoptotic bodies. Nat Genet 1998, 19:56–59.View ArticlePubMedGoogle Scholar
- Wessels MR, Butko P, Ma M, Warren HB, Lage AL, Carroll MC: Studies of group B streptococcal infection in mice deficient in complement component C3 or C4 demonstrate an essential role for complement in both innate and acquired immunity. Proc Natl Acad Sci U S A 1995, 92:11490–11494.View ArticlePubMedPubMed CentralGoogle Scholar
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