Various isoforms of laminins are present in the basement membranes complexed with
other structural proteins. In the central nervous system (CNS), they are primarily
localized to the basement membranes of blood vessels and reactive astrocytes. Limited
amounts of various laminin isoforms are also associated with neuronal cell bodies
and axons, which are referred to as “neuronal” laminins (Yamamoto et al., 1988). They
exist in a soluble form, free from other basement membrane proteins. Neurons either
produce these soluble laminins or acquire them from astroglial cells. Laminin stimulates
neuritogenesis and confers neuroprotection in vitro, but their exact role of these
neuronal soluble laminins in the CNS is yet unknown. Laminins bind to various cell-surface
receptors including integrins, dystroglycan, and the nonintegrin type 67 KDa laminin
receptor (67LR). In neurons, it is well known that 67LR internalizes prion proteins
and various bacteria and viruses (Nelson et al., 2008), but the signaling mechanisms
by which 67LR mediates neuroprotection, particularly by soluble laminin, is not clearly
known. Recently, we have found that soluble laminin (laminin-1 isoform) as well as
its YIGSR pentapeptide corresponding to the 67LR-binding sequence present in the β1-chain
of laminin, which can induce internalization of 67LR (Gopalakrishna et al., 2018).
This endocytosis is dependent on adenylyl cyclase, protein kinase A, and the exchange
protein directly activated by cyclic adenosine monophosphate (cAMP) (Epac). The internalized
endosomes comprising adenylyl cyclase and other signaling enzymes, continue to generate
signals such as cAMP for a sustained period of time which may contribute to neuroprotection.
Considering that β-amyloid peptide (Aβ) is internalized through 67LR (Da Costa Dias
et al., 2014), we postulate that laminin, by binding to and internalizing 67LR, inhibits
entry of Aβ into neurons and thereby decreases Aβ-induced neurotoxicity. Thus, laminin-related
agents that induce 67LR internalization could have therapeutic potential against Alzheimer’s
disease (AD) and various other neurodegenerative diseases.
Internalization of 67LR by soluble laminin and role of cAMP, protein kinase A, and
Epac: Our recent studies have shown that treatment of Neuroscreen-1 cells and its
parent PC12 cells with laminin-1 for a short period of time induced an internalization
of cell-surface associated 67LR (Gopalakrishna et al., 2018). 67LR-binding laminin
peptide YIGSR also induced internalization of cell-surface associated 67LR (
Figure 1
), whereas integrin-binding peptide GRGDS did not induce internalization of 67LR.
Cell-permeable analogs of cAMP also induced a decrease in cell-surface expression
of 67LR and induced its internalization, an effect mimicked by agents that elevate
intracellular cAMP, such as forskolin (which directly activates adenylyl cyclase and
thus elevates intracellular cAMP) and rolipram (which inhibits cyclic nucleotide phosphodiesterase
thus inhibits breakdown of cAMP). Consistent with this possible role of cAMP, laminin
or YIGSR failed to induce internalization of 67LR in PC12 cells deficient in protein
kinase A (PKA), suggesting the role of this kinase in the internalization. Another
downstream effector of cAMP, Epac, is also involved in 67LR internalization further
supporting the role of cAMP in this process.
Figure 1
Schematic presentation of possible neuroprotective action of laminin, its proteolytic
fragments, YIGSR peptide, and epigallocatechin-3-gallate (EGCG) via binding to 67
kDa laminin receptor (67LR) in opposition to β-amyloid peptide (Aβ).
Initial binding of laminin-1 and YIGSR to 67LR leads to activation of adenylyl cyclase
by an unknown mechanism. The resulting transient elevation of cyclic adenosine monophosphate
(cAMP), via activation of its effectors protein kinase A (PKA) and Epac, induces internalization
of lipid raft-associated 67LR. Along with 67LR, other lipid raft-associated signaling
enzymes, such as adenylyl cyclase, are also internalized. Early endosomes with activated
receptor complexes, adenylyl cyclase, and other signaling enzymes could serve as signalosomes
and induce a sustained elevation of cAMP and other signals. In addition, it is possible
that the early endosomes may recruit cell-survival enzymes and serve as signaling
platforms. Thus, the internalization of 67LR may confer neuroprotection. Similarly,
EGCG also binds to 67LR and induces cell signaling for neuroprotection. Both Aβ and
Aβ + prion complex bind to 67LR and their internalization causes toxicity. It is possible
that both laminin and EGCG may block the binding of Aβ to 67LR and prevent neuronal
toxicity induced by Aβ.
Colocalization of 67-LR and other lipid raft-associated enzymes to early endosomes:
Since 67LR and adenylyl cyclase are localized to lipid rafts, endocytosis of 67LR
by laminin and related agents could promote co-internalization of the some of the
lipid raft-associated signaling enzymes as well. Our study in fact, showed that 67LR
and adenylyl cyclase colocalize to early endosomes, suggesting that these early endosomes
may serve as “signalosomes” in neurons (Gopalakrishna et al., 2018). In this scenario.
endosome-associated adenylyl cyclase may contribute to a sustained generation of cAMP,
an important neuroprotective signal. Furthermore, these endosomes may recruit additional
cell-survival enzymes such as PI3 kinase, Akt, and PKC isoenzymes thereby serving
as robust signaling platforms for neuroprotection. Internalization of cell-surface
receptors has multiple consequences: the signal may be terminated; the receptor may
help in cargo delivery; receptor recycling may occur; and the receptor may be degraded
by lysosomes. In addition, early endosomes also function as signalosomes, causing
a sustained elevation of signals; this is shown in the case of parathyroid hormone,
which causes internalization of its own receptor along with adenylyl cyclase to early
endosomes, producing a sustained elevation of cAMP (Vilardaga et al., 2014).
Signaling associated with early endosomes may be very important, especially in neurons.
Internalization of nerve growth factor and brain-derived neurotrophic factor, along
with the appropriate receptors and signaling complexes, have been shown to play an
important role in further propagating the signal (Sorkin and von Zastrow, 2009). These
signalosomes are retrogradely transported from the distal axons to the soma to promote
transcriptional regulation. Previous studies showed that the laminin produced by some
neurons is taken up by other neurons and is retrogradely transported (Yamamoto et
al., 1988). This suggests that the internalization of laminin and its intracellular
transport may have a role in neuronal regulation. While these internalizations were
observed with laminin-1, whether other laminin isoforms also show this type of 67LR
internalization and cell signaling remains to be determined.
Significance of 67LR endocytosis to neuronal survival against neurotrophin deprivation:
A deprivation of neurotrophins leads to neuronal cell death. Currently, various studies
are being conducted to determine the efficacy of neurotrophins for treating various
neurodegenerative diseases. Cell death induced by serum deprivation of PC12 cells
is frequently used as a model for identifying neuroprotective agents and elucidating
their mechanisms. Using this model, we found the functional significance of the laminin
and its peptide YIGSR for neuroprotection. Laminin, YIGSR, dibutyryl cAMP, and forskolin,
all of which elevate intracellular cAMP, protected these cells from cell death induced
by serum deprivation. However, these agents protected wild-type PC12 cells having
PKA, they failed to protect PKA-deficient PC12 cells. Both adenylyl cyclase inhibitor
(SQ 22536) and Epac inhibitor (ESI-09) inhibited YIGSR-induced protection of Neuroscreen-1
cells from cell death induced by serum withdrawal. The conditions that induced endocytosis
of 67LR protected cells from death, whereas the conditions that did not induce the
internalization of 67LR did not protect cells from death. Thus, the internalization
of 67LR is important for laminin-mediated protection against cell death. The 67LR-blocking
antibody (MLuC5) suppressed neuroprotective effects of YIGSR peptide, suggesting the
role of this receptor in mediating neuroprotective action of this laminin peptide.
Certainly, additional studies, particularly in vivo, are warranted to further assess
the functional role of 67LR in neuroprotection.
Implication of 67LR internalization for protection against neurodegenerative diseases
such as AD: Recent studies have shown the role of 67LR in eliciting neurotoxicity
caused by Aβ, which is considered to play a crucial role in AD pathogenesis. Aβ binds
to 67LR either directly or indirectly through an initial association with prions that
subsequently bind to this receptor (Da Costa Dias et al., 2014). This leads to internalization
of 67LR and Aβ-mediated neurotoxicity. Although the exact site to which Aβ binds in
the 67LR sequence is not known, it is known that prions and YIGSR bind to the “peptide
G” sequence present within the 67LR. Therefore, laminin and YIGSR peptide could compete
with Aβ or prion-Aβ complex for 67LR and prevent their binding. In addition, the internalization
of 67LR caused by YIGSR may decrease the presence of 67LR on the cell surface for
the internalization of Aβ. Alternatively, the neuroprotective signaling induced by
laminin and YIGSR may protect from neurotoxicity induced by Aβ that enters the cell
through 67LR-independent mechanisms (Jarosz-Griffiths et al., 2016). For example,
Aβ signaling decreases the phosphorylation of cAMP response element-binding protein
(CREB), whereas laminin and other agents elevate cAMP, which could enhance the phosphorylation
of CREB and thereby provide a counteractive mechanism to overcome the toxicity induced
by Aβ. Previous studies have shown that laminin inhibits neuronal cell death by preventing
fibril formation and interaction of Aβ with cell membranes (Drouet et al., 1999).
It is also possible that a direct binding of laminin to cell-surface 67LR may be protective
against Aβ toxicity. Green tea polyphenols, such as epigallocatechin-3-gallate (EGCG),
have been shown to be neuroprotective in various neuronal diseases such as AD, Parkinson’s
disease, and stroke (Weinreb et al., 2004). Interestingly, EGCG binds with high affinity
to 67LR, induces internalization of this receptor, elicits neuroprotective signaling
and potentiates the action of neurotrophins (Tachibana et al., 2004; Gundimeda et
al., 2014). Since the EGCG-binding site on 67LR is in close proximity to the laminin-binding
site, it is possible that EGCG may counteract Aβ toxicity by mechanism(s) described
above for laminin.
There is an accumulating evidence that cerebrovascular injury/dysfunction represents
a major mechanism underlying neurodegeneration. Under this setting, vascular basement
membrane components are targeted for degradation by proteases such as metalloproteases
and cathepsins resulting in the release of soluble proteolytic fragments (Lee et al.,
2011). It is worth investigating whether the proteolytic fragments derived from laminin
diffuse into the brain parenchyma and promote 67LR-mediated signaling in neurons as
a protective response.
While 67LR is considered a culprit for cancer metastasis and internalization of pathogenic
prions, and certain bacteria and viruses (Nelson et al., 2008), it is also a receptor
for neuroprotective agents such as laminin, its peptides, and EGCG (Gundimeda et al.,
2014; Gopalakrishna et al., 2018). Its internalization by “pathogenic agents” could
lead to adverse events, but its internalization by “good agents” could lead to neuroprotection.
Thus, future understanding of the bidirectional role of this unique receptor may help
develop novel drugs for neuroprotection against AD, stroke and other neurodegenerative
conditions.