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.
KeywordsComplement Neurodegeneration Lysosomal storage disease Niemann-Pick Purkinje neurons Microglia 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.
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