Vol.2, No.4, 148-160 (2013) Advances in Alzheimer’s Disease
http://dx.doi.org/10.4236/aad.2013.24020
Accelerating Alzheimer’s pathogenesis by GRK5
deficiency via cholinergic dysfunction
William Z. Suo1,2,3
1Laboratory for Alzheimer’s Disease & Aging Research, Veteran Affairs Medical Center, Kansas City, USA;
2Department of Neurology, University of Kansas Medical Center, Kansas City, USA
3Department of Molecular & Integrative Physiology, University of Kansas Medical Center, Kansas City, USA
Received 26 September 2013; revised 5 November 2013; accepted 13 November 2013
Copyright © 2013 William Z. Suo. This is an open access article distributed under the Creative Commons Attribution License, which
permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
G protein-coupled receptors (GPCRs) mediate a
wide variety of physiological function. GPCR
signaling is negatively regulated by the receptor
desensitization, a procedure initiated by a group
of kinases, including GPCR kinases (GRKs). Stu-
dies using gene-targeted mice revealed that de-
ficiency of a particular GRK member led to dys-
function of a highly selectiv e group of GPCRs. In
particular, for example, GRK5 deficiency spe-
cifically disr upts M2/M4-mediated muscarin ic cho-
linergic function. Emerging evidence indicates
that ß-amyloid accumulation may lead to GRK5
deficiency, while the latter impairs desensitiza-
tion of M2/M4 receptors. Within memory circuits,
M2 is primarily presy napt ic autor ecep tor serv ing
as a negative feedback to inhibit acetylcholine
release. The impaired desensitization of M2 re-
ceptor by GRK5 deficiency leads to hyperactive
M2, which eventually suppresses acetylcholine
release and results in an overall cholinergic hy-
pofunctioning. Since the cholinergic hypofunc-
tioning is known to cause ß-amyloid accumula-
tion, the GRK5 deficiency appears to connect
the cholinergic hypofunctioning and ß-amyloid
accumulation together into a self-amplifying cy-
cle, which accelerates both changes. Given that
the ß-amyloid accumulation and the cholinergic
hypofucntioning are the hallmark changes in the
ß-amyloid hypothesis and the cholinergic hy-
pothesis, respectively, the GRK5 deficiency ap-
pears to bring the two major hypotheses in
Alzheimer’s disease t ogether, whereas the GRK5
deficiency is the pivot al link. Therefore, any stra-
tegies that can break this cycle would be thera-
peutically beneficial for Alzheimer’s patients.
Keywords: G Protein; Receptor; Kinase;
Cholinergic; Alzheimer; Pathogenesis
1. INTRODUCTION
Alzheimer’s disease (AD) affects 5.4 million Ameri-
cans, and is projected to double by 2020. AD is one of
the most persistent and devastating dementias with little
or no effective disease-modifying therapies. The cost of
care for this particular patient population is dispropor-
tionately high since they require expensive support. Ac-
cording to the Alzheimer’s Association, the cost of care
was $183 billion in 2010, which did not include 14.9
billion in services provided by unpaid caregivers. There-
fore, advances with translational potentials are highly
appreciable and desperately needed.
AD is a neurodegenerative disorder, clinically featured
with progressive loss of memory and other cognitive
functions, and pathologically featured with accumulation
of senile plaques (SPs) and neurofibrillary tangles (NFTs)
in limbic system and association cortices [1-4]. There
have been many hypotheses for AD, such as cholinergic
hypothesis, amyloid hypothesis, tau hypothesis, glucose
metabolism hypothesis, inflammatory hypothesis, vascu-
lar hypothesis, oxidative stress hypothesis, aluminium
hypothesis, and etc. [5-15]. It is indeed that each hypo-
thesis has its own supportive evidence and explains cer-
tain aspects of the disease process, yet none can see the
forest for the trees. Before a unifying hypothesis is born
and convincingly demonstrated, the field remains to be
diversified and appreciate any and all innovative ideas
with solid evidence. Numerous reviews have been writ-
ten to summarize advances in relation to the mainstream
hypotheses; this mini-review will instead briefly describe
recent progress related to deficiency of G protein-cou-
pled receptor (GPCR) kinase-5 (GRK5) in AD, and dis-

W. Z. Suo / Advances in Alzheimer’s Disease 2 (2013) 148- 160 149
cuss its relation to other hypotheses and the relevant fu-
ture perspectives.
2. CHARACTERISTICS OF GRK5
DISTRIBUTION AND FUNCTION
2.1. GRK5 and GRK Family
GRK is a small family (7 members) of serine/threon-
ine protein kinases first discovered through its role in
receptor desensitization [16-18]. GRK family members
can be subdivided into three main groups based on se-
quence homology: rhodopsin kinase or visual GRK sub-
family (GRK1/GRK7), the ß-adrenergic receptor kinases
subfamily (GRK2/GRK3) and the GRK4 subfamily
(GRK4/GRK5/GRK6). These kinases share certain char-
acteristics but are distinct enzymes with specific regula-
tory properties.
All GRK members contain a centrally located 263 -
266 amino acid (a.a.) catalytic domain flanked by large
amino- and carboxyl-terminal regulatory domains [19].
The amino-terminal domains share a common size (~185
a.a.) and demonstrate a fair degree of structural homol-
ogy. These characteristics have led to the speculation that
amino-terminal domains may perform a common func-
tion in all GRK members, potentially that of receptor
recognition. The primary function of GRK is to desensi-
tize activated GPCRs, a negative regulative process, in-
cluding phosphorylating the activated receptor, uncou-
pling the receptor-G-protein binding and initiating the
receptor internalization. GRK phosphorylates GPCR pri-
marily when the receptor is activated (agonist occu-
pied). The receptor phosphorylation triggers binding of
arrestins, which blocks the activation of G proteins,
leading to rapid homologous desensitization [19-21]. As
a result of the arrestin binding, the phosphorylated re-
ceptor is targeted for clathrin-mediated endocytosis, a
process that classically serves to resensitize and recycle
receptor back to the plasma membrane; or alternatively
sorts the receptor to degradation pathway [22].
2.2. GRK5 Expression and Distribution
GRK5 was originally cloned using polymerase chain
reaction (PCR) amplification of human heart and bovine
circumvallate papillae cDNA libraries with degenerate
oligonucleotide primers from highly conserved regions
unique to the GRK family [23,24]. Among 7 GRK mem-
bers, GRK5 belongs to those that are widely expressed in
various tissues, which is in contrast to GRK1, 4 and 7
that are confined to specific organs. For example, GRK5
mRNA is detectable in most tissues, whereas GRK1 and
7 are limited in retinal rods and cones, respectively, and
GRK4 is only present in testis, cerebellum and kidney
[23,24]. On the other hand, although GRK5 can be de-
tected in most tissues, its levels vary significantly, with
the highest levels in heart, lung, retina, placenta, and
moderate levels in skeletal muscle. Brain, liver, pancreas,
and kidney express very low levels of GRK5, with kid-
ney having the least.
As compared to cardiovascular tissues, GRK5 content
in brain is minimal [23,24]. This low content of GRK5 in
brain is in part because majority of the cortical areas has
little GRK5 expression, except for the limbic system [25].
The message of this kinase was found to be moderately
expressed in several limbic regions namely the cingulate
cortex, the septohippocampal nucleus, the anterior tha-
lamic nuclei, dentate gyrus of Ammon’s horn and the
medial habenula. Notably within these sub-regions that
express GRK5, the lateral septum was found to have the
highest GRK5 message. Therefore, this characteristic
distribution of GRK5 in brain discloses its unique func-
tional relation to the limbic system.
In addition to the tissue-specific distribution patterns,
increased GRK5 expression has been reported in relation
to over-expression of tazarotene-induced gene 1 (a tu-
mour suppressor gene) or α-synuclein, and in adriamycin
resistant tumor cells or hypothyroid animals, as well as
in AngII-treated vascular smooth muscle cells and ma-
crophage inflammatory protein-2 (MIP-2)-treated in po-
lymorphonuclear leukocytes (PMNs) [26-31]. In gen-
eral, there is a dearth of systematic studies that specifi-
cally address how GRK5 expression may be regulated
under different circumstances, including the normal re-
sponses to various physiological stimuli and possible
pathologic changes during aging and other disease con-
ditions, such as AD, drug abuse, cardiovascular disorders,
and cancers.
2.3. GRK5 Function
As a GRK family member, the primary function of
GRK5 is to desensitize activated GPCRs. Previous stud-
ies have shown that GRK5 regulates desensitization of
many GPCRs, including ß-adrenergic receptor (ßAR),
δ-opioid receptor (δ-OR), muscarinic receptors, angio-
tensin II Receptor (AngIIR), etc. [30,32-43]. Nonetheless,
increasing evidence indicates that GRK5 can also phos-
phorylate certain none-GPCR substrates (Table 1) and
thus affect their functions. For example, phosphorylation
of α-synuclein [44,45] and tubulin [46] by GRK5 has
been suggested to regulate their polymerization, and may
therefore be related to neuronal function and possibly
neurodegenerative disorders. Phosphorylation of p53 by
GRK5 regulates p53 degradation [47], which implies a
role of GRK5 in oncology. No matter if the substrates are
GPCRs, this part of GRK5 function requires its kinase
activity.
Beyond the kinase-dependent function, GRK5 has also

W. Z. Suo / Advances in Alzheimer’s Disease 2 (2013) 148- 160
150
Table 1. GRK5 substrates.
Name GPCR Origin Function Reference
AngIIR Yes Mouse AngIIR desensitization, and hypertension [30,32]
ßAR, ß1AR, ß2AR Yes Mouse Desensitization of ßAR (Gi), ß1AR, and ß2AR, related to
hypertension and heart failure [32-36]
δ-OR Yes In vitro/cells δ-OR desensitization and drug abuse [37]
FSHR Yes In vitro/cells FSHR desensitization and FSH signaling [52]
hSPR Yes In vitro hSPR desensitization [53]
M2 muscarinic
receptor Yes Mouse/airway smooth muscle/brain
M2 muscarinic receptor desensitization: asthma or
pulmonary disease, and AD [38-43]
PAR-1 Yes Endothelial cells PAR-1 desensitization [54]
TSHR Yes Thyroid cells TSHR desensitization [55,56]
a-synuclein No In vitro/cells/Lewy body a-synuclein oligomer formation and aggregation
in Parkinson’s disease? [44,45]
HDAC No Cardiomyocyte/mouse Myocardial hypertrophy [57]
Hip No In vitro/cells CXCR4 internalization [58]
NFkB p105 No Macrophages/ mouse LPS-induced inflammation/TLR4 signaling [59,60]
P53 No Osteosarcoma cells P53 degradation [47]
PDGFRß No In vitro PDGFRß desensitization [61,62]
Tubulin No In vitro/cells Tubulin dimmer assembling into microtubules [46]
Abbreviations: AngIIR, angiotensin II Receptor; ßAR, ß-adrenergic receptor; δ-OR, δ-opioid receptor; FSHR, follicle-stimulating hormone receptor; HDAC,
histone deacetylase; Hip, Hsp70 interacting protein; hSPR, human substance P receptor; PAR-1, thrombin receptor proteinase-activated receptor-1; PDGFRß,
platelet-derived growth factor receptor-ß; and TSHR, thyrotropin receptor.
been suggested to have kinase-independent regulatory
function. For example, GRK5 inhibits nuclear factor κB
(NFκB) transcriptional activity by inducing nuclear ac-
cumulation of IκB alpha [48,49]. GRK5 binds to Akt and
negatively regulates vascular endothelial growth factor
(VEGF) signaling [50]. Even more dramatically, GRK5
has been shown to contain a DNA-binding nuclear local-
ization sequence, and may possess potential nuclear trans-
criptional regulatory function [51]. Therefore, it appears
that GRK5 may have more divergent regulatory func-
tions than just being a GPCR kinase.
3. GRK5 DEFICIENCY AND ITS
PATHOLOGICAL CONSEQUENCES
3.1. GRK5 Deficiency
Although the dynamic regulation of GRK5 expression
remains to be systematically investigated, scattered re-
ports have indicated that GRK5 down-regulation may be
associated with prolonged platelet-derived growth factor
receptor-ß (PDGFRß) signaling, and treatments with
gonadotropin-releasing hormone (GnRH), thyroid stimu-
lating hormone (TSH), morphine, or lipopolysaccharide
(LPS) [31,55,62-64].
Even if the tGRK5 levels remain normal, the mem-
brane or functional GRK5 deficiency could occur due to
reduced membrane-associated GRK5 (mGRK5) levels
[40,65]. Given that desensitizing membrane-integrated
GPCRs is the primary function of GRK, the kinase itself
has to be physically associated with membrane to exe-
cute such a function. At physiological condition, GRK5
is primarily plasma membrane-associated by binding to
phosphatidylinositol-4, 5-bisphosphate (PIP2) and phos-
phoserine (PS), and is ready to act when GPCRs are ac-
tivated by their agonists [19,20]. In certain circumstances,
however, the balance of the binding force for GRK5 be-
tween membrane (e.g., PIP2) and cytosol (e.g., Ca2+/
Calmodulin) can be disrupted, which may cause translo-
cation mGRK5 to cytosol [65]. For example, in cultured
cells, Aß can cause rapid (within minutes) GRK5 mem-
brane disassociation and lead to functional mGRK5 defi-
ciency [40,66,67]. Therefore, GRK5 deficiency may be
caused by either decreased grk5 gene expression or re-
duced membrane distribution of the GRK5 protein, or
both.
3.2. Loss-of-Function in GRK5 Deficiency
As described above, accumulating evidence indicates
that GRK5 may have multiple regulatory roles in addi-
tion to primarily functioning as a GPCR kinase. Previous
studies have suggested that functional redundancy exists
between the seven GRK members in phosphorylating
different GPCR substrates. In fact, some may even sus-
pect that there might be little or no specificity among the
GRK members, mainly due the fact that seven or so

W. Z. Suo / Advances in Alzheimer’s Disease 2 (2013) 148- 160 151
GRK members are responsible for regulating hundreds to
thousands of GPCRs [20,65]. For the latter suspicion,
studies using GRK knockout (KO) mice have generated
unambiguous and convincing results that illustrate selec-
tive loss-of-function for each particular GRK member in
vivo. For example, the mice deficient in GRK2, 3, 5 and
6 display selectively impaired desensitization of adrener-
gic, odorant, muscarinic, and dopaminergic receptors,
respectively [38,68-70]. While recognizing the selective
loss-of-function, many of the known functions of a GRK
member are indeed not affected by the absence or defi-
ciency of this GRK member, which on the other hand
proves the redundancy or compensation between differ-
ent GRK members. In the case of GRK5 deficiency, for
example, the selective loss-of-function is evidenced only
for muscarinic, but not for adrenergic or opioid receptors
[38]. Nonetheless, many known GRK5 functions (i.e.,
those listed in Table 1) have not been specifically studied
to include/exclude as the part of functional loss in rela-
tion to GRK5 deficiency. Therefore, it is possible that
other functional loss caused by GRK5 deficiency, in ad-
ditional to the impaired muscarinic receptor desensitiza-
tion, remains to be discovered.
Interestingly, the loss-of-function caused by GRK5
deficiency on muscarinic receptor desensitization is re-
ceptor-subtype selective. To date, five muscarinic ace-
tylcholine (ACh) receptor subtypes have been identified,
with M1, M3, and M5 receptors being Gq/11-coupled, and
M2 and M4 receptors being Gi/o-coupled [71]. GRK5KO
mice, when challenged with non-selective muscarinic ago-
nists, display augmented hypothermia, hypoactivity,
tremor, and salivation, as well as antinociceptive changes
[38]. These behavioural changes are typical M2 and/or
M4 receptor-mediated functions, according to the find-
ings from muscarinic receptor subtype KO mice [71,72].
Therefore, although without molecular evidence, Gainet-
dinov et al. speculated in the original report that GRK5
deficiency primarily affected the desensitization of M2
subtype of muscarinic receptors (M2R), based on the
behavioural changes in the GRK5 KO (GRK5KO) mice
[38]. Going further, we demonstrated that GRK5 defi-
ciency led to reduced hippocampal ACh release that
could be fully restored by blocking presynaptic M2/M4
aotoreceptors [42]. Moreover, non-selective muscarinic
agonist-induced internalization of muscarinic receptors
was primarily impaired for M2R, partially for M4R, but
not for M1R. This study provided the molecular evidence
that confirmed the earlier speculation of the subtype-
selective effect of GRK5 deficiency on the inhibitory G
protein-coupled M2R and M4R, and revealed that the
immediate pathophysiological consequence of GRK5 defi-
ciency was the reduced ACh release (Figure 1).
3.3. Pathologic Impact of GRK5 Deficiency
Beyond the reduced ACh release, what pathologic im-
Figure 1. Schematic diagram of molecular interactions between
GRK5 and muscarinic receptors. The selective impact of GRK5
deficiency on presynaptic M2, but NOT postsynaptic M1, de-
termines its major impact is on the side of presynaptic cho-
linergic neurons, whereas its impact on the postsynaptic choli-
noceptive neurons is limited to the indirect effects of the re-
duced ACh release. For its presynaptic impact, GRK5 defi-
ciency leads to hyperactive M2 autoreceptor, which may not
only inhibit ACh release but also persistently suppress adenylyl
cyclase (AC) activity. The latter may impair the intrinsic de-
fense mechanisms and increase the cholinergic neuronal vul-
nerability to degeneration.
pact may GRK5 deficiency impose to AD? The evidence
in this line of work was initially obtained primarily from
GRK5KO mice.
We have mentioned that GRK5 may have multiple
functions that could be either kinase-dependent or inde-
pendent [65]. Of note, the GRK5KO mouse was created
by targeted deletion of exons 7 and 8 of the murine grk5
gene [38]. It was predicted that this mouse should pro-
duce a transcript encoding a peptide that contains 194
amino acid (a.a.). This speculative peptide should in-
clude the N-terminal a.a. 1 - 178 of the native GRK5
protein and an extra fragment of 16 novel residues. Gi-
ven that the RH domain of GRK5 locates at a.a. 50 - 176,
this means that the GRK5KO mice are only deficient in
the kinase-dependent function of GRK5, while the
kinase-independent or RH domain-dependent GRK5
function remains unaltered. In other words, the phenol-
types revealed in the GRK5KO mice are irrelevant to the
recently proposed RH domain-dependent GRK5 function
[48,49] or any other functions of GRK5 that are depend-
ent upon the N-terminal of 1 - 178 a.a. In addition, com-
pared to other AD mouse models, no genetic modifica-
tions were made to those commonly known AD-relevant
genes, such as ßAPP, presenilins, tau, or apolipoprotein
E, etc. Therefore, the phenotypes of these mice are solely
caused by the loss or lack of the GRK5 kinase activity.
The initial characterization of the GRK5KO mice re-
vealed that the young mice exhibited mild spontaneous
hypothermia as well as pronounced behavioural super-
sensitivity (i.e., hypothermia, hypoactivity, tremor, Sali-

W. Z. Suo / Advances in Alzheimer’s Disease 2 (2013) 148- 160
152
vation, and antinociception) upon challenge with the
non-selective muscarinic agonist oxotremorine [38]. Our
later characterization in the aged (18 months), unchal-
lenged mice discovered that the homozygous, but not the
heterozygous, GRK5KO mice displayed significant short-
term (working) memory deficit, along with hypo-alert-
ness [41].
At pathological level, the most prominent change was
the increased swollen axonal clusters (SACs) [41]. Inter-
estingly, this hallmark pathologic change in GRK5KO
mice primarily affected hippocampus. In the advanced
cases, it also affected the brain regions of piriform cortex,
amygdaloid and anterior olfactory nuclei, while most of
other brain regions were free of such pathological struc-
tures. We have mentioned that physiological GRK5
message expression in the brain is limited to the limbic
system [25]. Therefore, it seems that the sub-regions
where the SACs occur coincidently overlap with the
sub-regions where GRK5 message is enriched or where
these neurons are projected. It remains to be established
mechanistically that if such a coincidence underlies the
different aspects of the same mystery.
Beyond the axonal defects (SACs), we also found the
decreased synaptic proteins (SNAP-25, synaptotagmin,
and growth-associated protein-43) and muscarinic re-
ceptors (M1R and M2R), as well as increased soluble Aß
and tau phosphorylation levels in hippocampus of the
aged GRK5KO mice [41]. Moreover, the amnestic mild
cognitive impairment (MCI) in these mice was positively
correlated with the number of SACs and negatively cor-
related with the levels of M2R in the hippocampus, indi-
cating that there exist certain internal relations among
these changes in this animal model. It is worth noting
that there were barely any observable senile plaques (SPs)
or inflammatory changes, except for a few “micro” pla-
ques captured under electronic microscope that showed
fibrillar Aβ-like structures and degenerating axonal com-
ponents wrapped by reactive astrocytes. In particular the
lack of inflammation in this model was somewhat con-
trast to our earlier expectations, given that the initial in
vitro observation of Aß-induced GRK5 deficiency was
performed in microglial cells [40]. While analysing the
possible reasons, we attributed the negative inflamma-
tory reactions in this mouse to the fact that there was a
lack of significant extracellular Aß fibrils and/or any
other inflammatory initiators, whereas the GRK5 defi-
ciency can only play as an amplifier [41].
In order to verify these speculations, we crossbred the
GRK5KO mice with Tg2576 mice that over-express the
Swedish mutant human ßAPP [73]. The produced het-
erozygous GRK5 deficient APP mice are referred to as
GAP mice hereafter, although they were previously re-
ferred to as GRK5KO/APPsw double mice [43,74]. Stu-
dies in 18-month old GAP mice revealed significantly
exaggerated brain inflammatory changes, including mi-
crogliosis and astrogliosis, as compared to those in the
age-matched Tg2576 (APPsw) mice [74]. We have men-
tioned above that GRK5KO mice showed no inflamma-
tory changes [41], whereas the GAP mice displayed ex-
aggerated inflammation, much worse than Tg2576 mice.
As predicted earlier, the GRK5 deficiency indeed ampli-
fied the brain inflammation triggered by Aß fibrils that
are resulted from over-expression of the mutant human
APP.
In respect to the underlying mechanisms, we have
previously speculated that GRK5 deficiency may lead to
impaired desensitization of one or more GPCRs that are
involved in the fibrillar Aß-triggered inflammatory reac-
tions [65,74]. The latter could include a score of GPCRs,
such as formyl chemotactic receptor 2 (FPR2 or FPRL-1)
[75,76], C3aR and C5aR anaphylatoxin receptors [77],
and CCR, CXCR, and CXCXR chemokine receptors [78].
In addition, we did mention another possible explanation,
which was the increased Aß production itself [65]. Our
latest data indicated the latter predication (the increased
Aß rather than the impaired inflammatory GPCR desen-
sitization) is likely to be truth. First of all, none of the
aforementioned inflammatory GPCRs was found to be
affected by GRK5 deficiency. Secondly, we found that
not only soluble Aß production but also the fibrillar Aß
burden (both plaque number and area) were significantly
increased in the GAP mice. Moreover, the increased Aß
accumulation long preceded the exaggerated inflamma-
tion in the GAP mice [43]. In fact, our further analysis of
the pooled data revealed that there exist strong positive
correlations between the fibrillar Aß burden and the glio-
sis in the GAP mice (Figure 2). Therefore, it becomes
clear now that the exaggerated inflammation in the GAP
mice occurs only after and is a consequence of the in-
creased fibrillar Aß deposits.
Figure 2. Correlation between Aß+ plaque burden and gliosis in
the GAP mice. Significant linear correlations of Aß+ plaque
area burden with CD45+ microglial (A) and GFAP+ astrocyte
(B) cell numbers in hippocampus of the GAP mice were shown
as indicated. The analyses used pooled data from 18 months old
female GAP and control Tg2576 mice with a sample number
≥12.

W. Z. Suo / Advances in Alzheimer’s Disease 2 (2013) 148- 160 153
4. MECHANISTIC LINKS BETWEEN
MOLECULAR EVENTS DRIVEN BY
GRK5 DEFICIENCY
4.1. Cholinergic Dysfunction and
ß-Amyloidogenesis
The pathologic impact of GRK5 deficiency in the
GAP mice has now come to a simple point: the increased
Aß production; whereas the immediate pathophysiologi-
cal consequence of GRK5 deficiency is reduced ACh
release [42]. Is there an internal relation between these
changes? In this regard, our latest study untangled their
relations [43]. We found that GRK5 deficiency altered
ßAPP processing in favour of ß-amyloidogenic pathway,
which was mediated by the impaired cholinergic activity.
From the perspective of phenomenon, we have ob-
served and reported previously in the GRK5KO mice
that the GRK5 deficiency promoted soluble Aß accumu-
lation [41]. Perhaps because that it was murine Aß, we
failed to observe significant fibrillar Aß deposit in the
GRK5KO mice. In the GAP mice, however, we found
excessive Aß accumulation, not only the soluble form,
but also the fibrils deposited in the plaques. Compara-
tively, the total Aß burden in the GAP mice at 18 month
old was roughly doubled than that in Tg2576 mice at the
same age [43]. Therefore, there is no question that GRK5
deficiency promotes Aß accumulation. As for the rele-
vant mechanism, we demonstrated that there was in-
creased secreted APPß fragment without change of the
full length APP in the GAP mice, indicating there was an
increased ß-amyloidogenic APP processing in this ani-
mal model. In an acute experimental model, moreover,
we also measured the dynamic change of interstitial fluid
(ISF) Aß in hippocampus of the mice. We found that the
ISF Aß levels decreased when Tg2576 mice were chal-
lenged with novel object introduction (NOI) while this
ability was retarded in the GAP mice. What interesting
was that this difference between the GAP and Tg2675
mice was completely corrected by selective M2 antago-
nization, which clearly linked the increased Aß produc-
tion in the GAP mice to a cholinergic dysfunction, or
more specifically to the presynaptic M2 hyperactivity
that we previously described [43,65].
The impaired desensitization of M2R is so far the only
demonstrated molecular functional loss after GRK5 defi-
ciency [38,42]. It is known that M1, M2 and M4, but not
M3 and M5, are enriched in hippocampus, with M2/M4
being primarily presynaptic autoreceptors to negatively
regulate ACh release in hippocampal memory circuits
[79,80]. Therefore, once presynaptic M2R or M4R is
activated, it is conceivable that the M2/M4 signalling
will be prolonged, if there is GRK5 deficiency. This
prolonged presynaptic M2/M4 autoreceptor signalling
was referred to as presynaptic cholinergic hyperactivity
[42,43,65]. The best-known effect of the presynaptic
cholinergic hyperactivity is the reduced ACh release [42],
which in turn leads to postsynaptic cholinergic hypoac-
tivity, including postsynaptic M1 hypoactivity. In fact,
postsynaptic cholinergic hypoactivity (reduced ACh) has
been previously shown to promote the ß-amyloidogenic
APP processing and Aß production [81-85]. Therefore, it
is no surprise that blocking the presynaptic M2R and the
prolonged M2 signalling completely restored the ability
for the GAP mice to down-regulate the ISF Aß produc-
tion.
Aside from the increased Aß accumulation and the
subsequently exaggerated inflammation, another promi-
nent pathologic change associated with GRK5 deficiency
is the axonal defects and the reduced synaptic proteins
and muscarinic receptors [41]. It remains to be estab-
lished that whether the observed axonal defects and syn-
aptic degenerative changes are cholinergic and/or choli-
noceptive selective. Should it be the case, which is likely,
the axonal defects and synaptic degenerative changes
could be closely related pathologic events. There is no
direct evidence suggesting that these pathologic changes
are resulted from the presynaptic cholinergic hyperactiv-
ity, but supportive evidence certainly exists. For example,
previous studies suggested that signaling of M1, M3 and
M5, but not M2 or M4, appears to be anti-apoptotic [86].
M1 signaling was shown not only to inhibit ß-amyloi-
dogenic APP processing but also to decrease tau phos-
phorylation in vitro [81,87,88]. Therefore, M1 signalling
is generally characterized as “cholinergic protective”. On
the other hand, M1 and M2 are typical Gq and
Gi-coupled receptors, respectively, and often mediate
distinct or even opposing signals [89,90]. M2 signalling
is known to reduce cAMP level [89,90], and
down-regulate protein kinase A (PKA) activity, a vital
signalling pathway for cell survival and apoptotic resis-
tance [91-96]. Maybe more relevant to axonal defects
and synaptic degeneration, PKA phosphorylates and in-
activates glycogen synthase kinase 3 (GSK3) to facilitate
glucose metabolism and cell/neurite growth [97-100]. In
the case of M2 hyperactivity, PKA will be persistently
inhibited. In this case, GSK3 will be released from their
complex, and become dephosphorylated and activated.
The latter, especially GSK3ß, which is also known as tau
protein kinase-1 (TPK1), is one main cause of tau hy-
perphosphorylation [101]. Tau hyperphosphorylation
destabilizes microtubules and renders itself more prone
to aggregation [102,103], which can contribute to axonal
defects. Moreover, GSK3ß can also phosphorylate kine-
sin light chain (KLC), which leads to detachment of the
kinesin motor from the cargo, thus preventing further
transport of cargo and resulting in axonal swellings
[102,104,105]. Therefore, if M1 signalling is “choliner-
gic protective”, then M2 signalling appears to be “cho-

W. Z. Suo / Advances in Alzheimer’s Disease 2 (2013) 148- 160
154
linergic destructive”, at least when it is prolonged. In the
case of GRK5 deficiency, both presynaptic M2 hyperac-
tivity (destructive) and postsynaptic M2 hypoactivity
(less protective) would be detrimental to the cholinergic
neuronal system (see Figure 3 for schematic illustration
of the hypothesis).
4.2. GRK5 Deficiency Links the Amyloid and
Cholinergic Hypotheses Together
Despite various views on detailed causes and proc-
esses of AD, two major hypotheses have driven pharma-
ceutical research of AD in recent three decades: the
amyloid hypothesis and the cholinergic hypothesis [106].
The cholinergic hypothesis states that central cholinergic
neuronal dysfunction is largely responsible for the cogni-
tive decline in AD [5]. The amyloid hypothesis proposes
that Aß is the central pathogenic molecule in AD [6].
Although the details of these two hypotheses may evolve
over time with increasing insights into disease patho-
genesis, the principal concepts of these distinct hypothe-
ses appear to stand solidly [6,106-112].
First, Aß is one of the main causes for mGRK5 defi-
ciency in AD [40]. Once GRK5 is deficient, it selectively
impairs desensitization of presynaptic M2/M4 autore-
ceptors, which leads to presynaptic cholinergic hyperac-
tivity and the subsequent postsynaptic cholinergic hypo-
activity. The latter, as the key component of the cho-
linergic hypothesis, further accelerates Aß production.
Therefore, these studies directly connect Aß → GRK5
deficiency → cholinergic dysfunction → Aß into a self-
promoting loop or vicious cycle. In this vicious cycle, Aß
and cholinergic dysfunction each can serve as the causes
Figure 3. Schematic illustration of
relations of GRK5 deficiency to the
ß-amyloid and the cholinergic hy-
potheses. BFC, basal forebrain cho-
linergic.
and consequences, while GRK5 deficiency is the pivotal
mediator (Figure 3). Given the dominating importance
of both amyloid and cholinergic hypotheses in AD, more
efforts should be directed to studies of GRK5 deficiency
that has been overlooked in the past.
It is worth noting that cholinergic dysfunction is a
relatively broad term describing the deficiency of neuro-
transmitter ACh at cholinergic terminals. Cholinergic
dysfunction in AD, according to the cholinergic hypo-
thesis, is characterized by cholinergic hypofunction or a
reduction of ACh, which may result from known changes
in AD brains, such as reduced choline acetyltransferase
(ChAT) and choline uptake, cholinergic neuronal and
axonal abnormalities, and degeneration of cholinergic
neurons [5,110]. These pathological characteristics of
AD observed from postmortem brain tissues of AD pa-
tients are evident changes at very late stages of the dis-
ease. Some late stage changes may not necessarily re-
veal or reflect more causative alterations that occur early
in the disease process. For example, activity of choliner-
gic markers, such as ChAT and acetylcholinesterase
(AChE), do not decrease until very late stages of the dis-
ease [113]. Since neither ChAT nor AChE is ratelimiting,
changes in these markers do not necessarily reflect cho-
linergic function [114]. In fact, ChAT can be inhibited up
to 90% with no measurable effects on ACh synthesis or
release [107], while pharmacological and neurophysi-
ological deficits in cholinergic response can exist without
significant changes in ChAT activity during normal ag-
ing [114]. In line with the latter situation, we have pre-
viously shown that cholinergic hypofunction (reduced
ACh release) can exist in the absence of cholinergic
structural degeneration in young GRK5KO mice [42],
and this only turns into a cholinergic dysfunction with
structural degenerative changes in aged GRK5KO mice
[41]. We have thus questioned whether early nonstruc-
tural cholinergic dysfunction that precedes the struc-
tural cholinergic dysfunction/degeneration could also be
causative for the latter [42].
While recognizing the importance of the GRK5 defi-
ciency in AD, many important questions remain to be
specifically addressed. Based on existing literature in-
formation and our own evidence, we present a hypothetic
model that links GRK5 deficiency to the amyloid and
cholinergic hypotheses in AD (Figure 3). This hypo-
thetic model attempts to integrate the two most important
hypotheses in AD into one unifying hypothesis, in hope
to encourage more investigations for validations and for
ultimately improving our understanding of the disease
pathogenesis. From therapeutic perspective, moreover, it
emphasizes the presynaptic autoreceptors as the key tar-
get for breaking the vicious cycle between Aß and cho-
linergic dysfunction.

W. Z. Suo / Advances in Alzheimer’s Disease 2 (2013) 148- 160 155
5. CONCLUSION
As described, GRK5 deficiency emerges to become a
critical player in AD pathogenesis, not only due to its
close relation to Aß, but also because of its selective ef-
fect on cholinergic dysfunction. Just like Parkinson’s
disease is marked with dopaminergic neurodegeneration,
AD as a neurodegenerative disorder is labelled with cho-
linergic selective neurodegeneration. Yet, neurotoxic fac-
tors identified as far, including Aß, are not necessarily
more toxic to cholinergic neurons than to non-cholinergic
neurons; whereas all the non-selective neurodegenerative
forces (i.e., Aß, oxidative and inflammatory damages) in
AD convert to cholinergic selective neurotoxicity is like-
ly determined by the GRK5 deficiency. For proof-of-
the-principle, the immediate future effort should address
whether or not blocking the presynaptic M2 hyperactiv-
ity abolishes all the pathologic events driven by the
GRK5 deficiency.
6. ACKNOWLEDGEMENTS
This work was supported by grants to W.Z.S. from the Medical Re-
search and Development Service, Department of Veterans Affairs, the
Alzheimer’s Association, and resources from the Midwest Biomedical
Research Foundation.
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