Keystone: Probing the Function of Lipoprotein and Related Receptors

Series - Keystone: Slalom No Match for Ins and Outs of ApoE Research:

On the face of it, lipoprotein receptors may not sound like they have much to do with Alzheimer’s disease, or even the central nervous system. But as scientists are finding out more about these multifaceted cell-surface proteins, they are discovering just how intimately involved they are in the care and maintenance of neurons and their synapses. At "ApoE, Alzheimer’s and Lipoprotein Biology," a Keystone symposium held 26 February-2 March, 2012, presentations reflected the breadth and depth of the biology of these receptors. The conference drove home to attendees how the receptors’ functions dovetail with neurobiology and, potentially, neurodegeneration.

One member of the low-density lipoprotein receptor family that is familiar to AD researchers is SorLA (short for the unfortunate mouthful, sortilin-related receptor, low-density lipoprotein receptor class A repeat-containing protein). In the last decade, researchers, including those in Thomas Willnow’s lab at the Max Delbruck Center for Molecular Medicine, Berlin, Germany, discovered that SorLA (aka SORL1 and LR11) regulates processing of amyloid-β (Aβ) precursor protein (see ARF related conference story). Variants in the SorLA gene subsequently emerged as risk factors for late-onset AD (see ARF related news story). Using overexpression and knockout models, researchers gradually built a picture of the protein sequestering APP and keeping it away from endosomes, where β- and γ-secretases would process it to release Aβ. That is the simple view, Willnow said at the Keystone symposium. In fact, he said, exactly how SorLA regulates APP processing is still being worked out. The overexpression and knockout models are probably too drastic to reflect what happens in a physiological setting. In a talk that stood out for detailing a rigorous biochemical approach, according to meeting co-organizer Joachim Herz of the University of Texas Southwestern Medical Center, Dallas, Willnow reported how even slight tweaks in levels of APP and/or SorLA profoundly affect their choreography, suggesting that modest changes in levels of SorLA may be meaningful in AD.

Willnow used the tetracycline (tet-off) system for controlling gene expression to alter transcription of both SorLA and APP by small increments in a cell-based system. He mathematically modeled the relationship between the two proteins and the production of sAPPα and sAPPβ. His data basically boiled down to a major kinetic finding, namely, APP processing in the absence of SorLA does not follow Michaelis-Menten kinetics. For those who remember their biochemistry, that predicts a simple enzyme-cleaves-substrate type of reaction. Instead, the data fit Hill kinetics, which assumes cooperativity between APP molecules, said Willnow. In fact, the Hill coefficient for APP processing is 2.0, which implies that secretases preferably process APP as a dimer. In the presence of SorLA, the coefficient reverts to 1.0, indicating non-cooperativity and APP monomer processing.

How could SorLA alter kinetics? Willnow’s data indicate the lipoprotein receptor and APP together form a dimer, and SorLA prevents APP dimerizing with itself, at least in Chinese hamster ovary cells. Western blots revealed an APP dimer on native gels, which disappeared upon coexpression of SorLA. Mouse brain showed a similar pattern, whereby extracts from wild-type mice contained big and small APP species, but extracts from SorLA knockouts only the larger.

“This work really takes us back to basic principles and gives us the molecular details we need to understand how these receptors work,” Herz told Alzforum after Willnow’s talk. The kinetic data are particularly relevant to normal physiology, said Willnow, because if there is cooperativity in processing, then a small change in APP concentration can have a large change on Aβ production. It could also explain how non-coding genetic variants that modestly perturb SorLA expression might affect risk for dementia. Researchers have been struggling to interpret how non-coding genetic variants uncovered by genomewide association studies alter risk.

Not just SorLA, its relatives, too, came up for discussion at the symposium. SorLA is part of a family of Vps10p domain receptors (named after the vacuolar protein sorting 10 protein domain that they all share) that also contains SorCS isoforms and sortilin. Anders Nykjaer, Aarhus University, Denmark, together with Stephen Strittmatter at Yale University, New Haven, Connecticut, found that sortilin binds progranulin and carries it to the lysosome for degradation (see ARF related news story on Hu et al., 2010). Genetic variants near the sortilin gene are also risk factors for frontotemporal lobar degeneration (FTLD) (Carrasquillo et al., 2010). Reducing sortilin could, therefore, elevate levels of progranulin, which is essential to stave off FTLD (see ARF related news story). But as Nykjaer explained in his talk, the picture is not so simple.

Nykjaer and colleagues found that sortilin regulates the balance between long-term potentiation (LTP), and long-term depression (LTD) because it controls levels of brain-derived nerve factor (BDNF). Working in cooperation with tyrosine receptor kinase B (TrkB), BDNF promotes LTP, while the immature proBDNF, working through p75, induces LTD (see ARF related news story on Woo et al., 2005). Nykjaer showed that sortilin stabilizes proBDNF, and that without the receptor, proBDNF quickly degrades and LTD dwindles, while short-term LTP escapes unscathed. However, late-phase LTP, which depends on localized conversion of proBDNF to BDNF at synapses, is weakened in sortilin knockouts.

What could this mean for synaptic activity in a physiological setting? Nykjaer showed that mice without sortilin have behavioral problems. They respond to some environmental challenges in a similar fashion to people who suffer from bipolar disorder or schizophrenia, said Nykjaer, and in Denmark, genetic screens uncovered single nucleotide polymorphisms that are linked to such disorders.

Neuromuscular Junction as a Model System
Steve Burden, New York University, also addressed synaptic roles for lipoprotein receptors. Burden looked to the neuromuscular junction (NMJ) to identify functions for these receptors, and he emphasized that some of the same molecules once thought to exist solely at NMJs have since been discovered in synapses in the central nervous system. Several researchers at the meeting agreed that looking at NMJs may help scientists understand synapse maintenance and loss in the CNS. This is germane to Alzheimer’s pathology, he noted, in the sense that the disease is widely believed to be one of synaptic failure. Lipoprotein receptors turn out to be essential for the maintenance of NMJs, raising the possibility that they may also be indispensible for synapses in the brain.

Burden’s talk focused on low-density lipoprotein receptor-related protein 4 (Lrp4), which coordinates the clustering of acetylcholine (ACh) receptors on the muscle side of the neuromuscular synapse. Burden noted that Agrin, a protein released by motor neuron axons, binds Lrp4. The lipoprotein receptor in turn activates MuSK kinase, setting off a signaling cascade that upregulates expression of ACh receptor genes on the post-synaptic side of the junction. This cascade plays out as developing motor neurons seek out muscle tissue to innervate. Early in development, Lrp4 binds and activates MuSK independently of Agrin, reported Burden. This sets up an initial incorporation of ACh receptors into the muscle in anticipation of the arrival of the motor neuron. Agrin then boosts the Lrp4-MuSK interaction 50-fold, stabilizing the NMJs. Burden’s lab found that only the ectodomain of Lrp4 is essential for these interactions. He rescued the Agrin response in Lrp4-negative cells by simply expressing this external domain of Lrp4, or a chimera with the intracellular domain substituted with one from the CD4 receptor (see Gomez and Burden, 2011).

So far so good—but what happens on the motor neuron side of the equation? That is mostly unknown, said Burden. Without MuSK or Lrp4, motor neuron axons do not stop when they reach muscle cells, but keep growing around and pass them. Burden wondered if Lrp4 corrects this by activating MuSK and setting off signals solely within the muscle tissue, or if it somehow signals directly to the developing axon. To test this, Burden and colleagues co-cultured motor neurons with fibroblasts engineered to express Lrp4. Lo and behold, these cells induced clustering of presynaptic vesicle and active zone proteins in the motor neuron axons. Lrp4-coated beads did, too. The scientists found that the Lrp4 ectodomain binds to motor neurons, supporting the idea that the lipoprotein receptor directly signals the cells. He concluded that the lipoprotein receptor controls both the muscle and the neuron side of the developing NMJ. The related Lrp1 and Lrp6 had no such effects, suggesting the property may be unique to Lrp4.

How could this be relevant to the brain, or to AD? CNS expresses Agrin, Lrp4, and MuSK, noted Burden, and their roles there are unclear. But given that Agrin prevents synapse loss in the cortex (see Ksiazek et al., 2007), its signaling might be relevant not just to the neuromuscular system, but also to neurodegenerative disease.—Tom Fagan.

This is Part 2 of a five-part story. See also Part 1, Part 3, Part 4, Part 5. Download a PDF of the entire series.

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References

News Citations

  1. Sorrento: Sorting Out Shedding of Ectodomains
  2. SORLA Soars—Large Study Links Gene to Late-onset AD
  3. Sorting Progranulin With Sortilin—New Clues to FTLD Pathology
  4. Birds of a Feather…Mutations in Tau Gene Neighbor Progranulin Cause FTD
  5. What Role BDNF?—A Question of Maturity
  6. Keystone: Symposium Emphasizes Key Aspects of ApoE Biology
  7. Keystone: ApoE Receptors and Ligands in Memory and AD
  8. Keystone: Does ApoE Fragmentation Drive Pathology?
  9. Keystone: Therapies Around ApoE—Has Their Time Come?

Paper Citations

  1. Hu F, Padukkavidana T, Vægter CB, Brady OA, Zheng Y, Mackenzie IR, Feldman HH, Nykjaer A, Strittmatter SM. Sortilin-mediated endocytosis determines levels of the frontotemporal dementia protein, progranulin. Neuron. 2010 Nov 18;68(4):654-67. PubMed.
  2. Carrasquillo MM, Nicholson AM, Finch N, Gibbs JR, Baker M, Rutherford NJ, Hunter TA, Dejesus-Hernandez M, Bisceglio GD, Mackenzie IR, Singleton A, Cookson MR, Crook JE, Dillman A, Hernandez D, Petersen RC, Graff-Radford NR, Younkin SG, Rademakers R. Genome-wide screen identifies rs646776 near sortilin as a regulator of progranulin levels in human plasma. Am J Hum Genet. 2010 Dec 10;87(6):890-7. PubMed.
  3. Woo NH, Teng HK, Siao CJ, Chiaruttini C, Pang PT, Milner TA, Hempstead BL, Lu B. Activation of p75NTR by proBDNF facilitates hippocampal long-term depression. Nat Neurosci. 2005 Aug;8(8):1069-77. PubMed.
  4. Gomez AM, Burden SJ. The extracellular region of Lrp4 is sufficient to mediate neuromuscular synapse formation. Dev Dyn. 2011 Dec;240(12):2626-33. PubMed.
  5. Ksiazek I, Burkhardt C, Lin S, Seddik R, Maj M, Bezakova G, Jucker M, Arber S, Caroni P, Sanes JR, Bettler B, Ruegg MA. Synapse loss in cortex of agrin-deficient mice after genetic rescue of perinatal death. J Neurosci. 2007 Jul 4;27(27):7183-95. PubMed.

Other Citations

  1. Download a PDF of the entire series.

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