Like overzealous gardeners, microglia have been found to cut back synapses in mouse models of Alzheimer’s and Huntington’s disease. Now, researchers led by Eric Huang at the University of California, San Francisco, report that a similar process may occur in mice that lack progranulin. Deficiency of this gene causes as many as 20 percent of familial FTD cases. In progranulin knockout mice, microglial activity ramped up. These immune cells overproduced complement proteins, which tagged synapses for destruction, while also dialing up lysosomal digestion. As a result, these microglia devoured more synapses than did microglia in control brains. “This study provides additional evidence that aberrant activation of microglia can indeed drive neurodegeneration,” Huang told Alzforum.
The findings may be applicable to human disease, the authors note, because autopsy data from people with this form of FTD revealed excessive complement-labeled synapses in the frontal cortex. In addition, increased cerebrospinal fluid levels of the complement proteins C1q and C3 correlated with cognitive decline in these patients, Huang and colleagues found. The finding hints that these proteins could have potential as biomarkers of disease progression, Huang noted.
Jonathan Kipnis at the University of Virginia, Charlottesville, agreed the data add to an emerging picture that puts microglia at the center of synaptic loss in multiple disorders. “I think it is clear now that microglia and their phagocytic activity could be real [therapeutic] targets in neurodevelopmental and also in late-stage neurodegenerative diseases,” he wrote to Alzforum.
In the past decade, researchers including Beth Stevens at Boston Children’s Hospital have developed the idea that microglia and astrocytes are responsible for the synaptic pruning that occurs as the mammalian brain develops (see Dec 2000 news; Nov 2007 conference news; Mar 2015 conference news). Recently, multiple groups extended these findings to disease states, reporting that this pruning pathway reactivates in some neurodegenerative disorders and even in conditions such as obesity (see Aug 2013 conference news; Dec 2014 news; Nov 2015 conference news).
Huang and colleagues wondered if the same thing might happen in progranulin-deficient animals. After all, microglia produce most of the progranulin in the brain (see Zhang et al., 2014). Previous studies by the authors and others had seen neuroinflammation and microglial activation in the absence of this protein, suggesting progranulin normally suppresses these cells (see Yin et al., 2010; Mar 2011 news; Oct 2012 news). However, the mechanisms were unclear.
To pin down what progranulin might be doing, first author Hansen Lui, now at the University of California, Berkeley, compared gene expression in the cerebral cortex, hippocampus, and cerebellum of progranulin knockout mice (GRN-/-) to that of wild-type mice at various ages. Progranulin knockouts develop neuroinflammation and lose inhibitory synapses as they age. Their neuronal circuits become overly excitable, and they groom themselves excessively. Compulsive behaviors mark FTD as well. GRN-/- mice die about 200 days sooner than wild-types. Lui and colleagues found that with age, GRN-/- microglia overexpressed genes involved in innate immunity and lysosomal digestion. The data implied that these two processes played a key role in pathogenesis.
To confirm this, the authors examined mouse brains and found that at 16 months of age, GRN knockouts had four times the normal amount of microglia. These cells contained enlarged lysosomes, suggesting digestion of cellular waste might be perturbed. In keeping with this, GRN-/- microglia from older mice ingested and processed a fluorescent dye more quickly in culture than wild-type microglia did. Likewise, in co-cultures with neurons, the GRN-/- microglia gobbled more synapses, accumulating about 60 percent more synaptic markers than did normal microglia.
In addition to the lysosomal changes, microglia from frontal cortex, hippocampus, and other regions of older GRN-/- mice produced more complement than did microglia from wild-types. The change was most pronounced in the thalamus, where GRN knockout mice pumped out up to eight times more C1q and 100-fold more C3 at 18 months of age. All these regions are sites of pathology in FTD as well. The authors focused on the thalamus in subsequent experiments. They found that thalamic synapses sported increasing amounts of these complement proteins as the mice aged. Moreover, GRN knockout microglia were overeager in lysosomal processing of C3, a process that releases active fragments of this complement factor, including iC3b. GRN-/- microglia made four times as much iC3b as wild-types. This is likely due to faster lysosomal digestion in the knockouts, Huang said. The lysosomal defect might boost the innate immune system as well as stimulating digestion of synapses, he suggested.
“The two phenotypes together create a perfect storm,” Huang told Alzforum.
Do these changes in complement and lysosomes affect synapse loss directly in vivo? To find out, the authors crossed C1q knockouts with GRN-/- mice. In contrast to the latter, which had lost a third of their synapses by 19 months of age, the double knockouts maintained normal synaptic density in the thalamus even past that age (see image above). The lack of complement also improved neuronal function and the overall health of the animals. Brain slices from double knockouts fired normally. They lived about 100 days longer than the GRN-/- mice and groomed no more often than wild-type animals.
Preliminary data tie these findings to FTD pathology. The authors examined autopsy samples from the frontal cortices of 19 patients with GRN mutations and found up to fourfold higher microglial density compared to controls, and profuse C1q at synapses. In cerebrospinal fluid, absolute levels of complement proteins did not differ between FTD patients and controls, however, the amount of C1q and C3 in patient CSF climbed as cognition faltered. If that relationship holds in larger samples, complement proteins could become biomarkers of disease progression or therapeutic benefit, Huang suggested.
The findings distinguish FTD from AD. Complement has been linked to synapse loss in Alzheimer’s disease (see Apr 2016 news), but the authors found a different pattern of neuroinflammation and biomarkers in the two disorders, suggesting distinct mechanisms might be at work. In AD brains, microglial density was similar to that in controls, and most of the immune cells clustered around plaques. In AD CSF, C1q levels were lower than in controls, and kept going down. Huang noted that complement proteins deposit in amyloid plaques, perhaps explaining their scarcity in CSF just as Aβ42 goes down in AD CSF. In addition, Huang suggested that complement factors in AD and progranulin-deficient FTD might come from different sources. In AD brains, microglia do not appear to overproduce complement, rather, it may come from astrocytes or the bloodstream, Huang speculated. In future work, he plans to look for elevated complement in patient CSF and brain tissue samples from other subtypes of FTD.
Many other questions remain. In GRN-/- mice, the authors found C1q equally distributed among excitatory and inhibitory synapses but, curiously, microglia only pruned the latter. Other signals present at the synapse might protect excitatory synapses, Huang suggested. Huang will examine conditional GRN-/- mice that lack the protein only in microglia or only in neurons, and he intends to test a C1q-blocking antibody from Annexon Biosciences, South San Francisco, to see if the treatment can preserve synapses and improve behavior. Other researchers noted that the links between lysosomal defects and the upregulation of complement proteins are unclear and deserve further investigation. Yoshinori Tanaka at the Tokyo Metropolitan Institute of Medical Science pointed out that C1q production also rises in some lysosomal storage diseases (see Ohmi et al., 2003).
Huang also wants to follow up on the lysosomal phenotype and dissect how progranulin regulates lysosomal digestion. He suspects he will find links between progranulin-deficient forms of FTD and autoimmune disease. Enhanced processing of C3 by lysosomes in T cells has been found to worsen inflammation in people with autoimmune arthritis (see Liszewski et al., 2013). Moreover, FTD patients have an increased incidence of autoimmune disorders. Alzheimer’s disease also was recently reported to share genetic risk factors with some autoimmune conditions (see Apr 2016 news).
Tim Sargeant at the South Australian Health & Medical Research Institute, Adelaide, pointed out that complete loss of progranulin causes the lysosomal storage disease neuronal ceroid lipofuscinosis. Loss of several other proteins produce similar lysosomal defects and neurodegeneration, he added. “It is therefore possible the effects observed in this paper are from a lysosomal defect that can be caused by deficits in a wide range of lysosomal proteins. These observations … show targeting of lysosomal function in late onset neurodegenerative disease could offer a feasible therapeutic strategy,” he wrote to Alzforum (see full comment below).—Madolyn Bowman Rogers
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