Keystone: Probing the Function of Lipoprotein and Related Receptors

This is Part 2 of a five-part story. See also Part 1


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

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.

This is Part 2 of a five-part story. See also Part 1.



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