Associate editor: C. Stevenson
GDNF, NGF and BDNF as therapeutic options for neurodegeneration
Shelley J. Allena,⁎, Judy J. Watsona, Deborah K. Shoemarka, Neil U. Baruab, Nikunj K. Patelb
aDorothy Hodgkin Building, Whitson St, Bristol BS1 3NY, UK
bDept of Neurosurgery Frenchay Hospital, Bristol BS16 1LE, UK
a b s t r a c ta r t i c l ei n f o
Glial cell-derived neurotrophic factor (GDNF), and the neurotrophin nerve growth factor (NGF) and
brain-derived neurotrophic factor (BDNF) are important for the survival, maintenance and regeneration of
specific neuronal populations in the adult brain. Depletion of these neurotrophic factors has been linked
with disease pathology and symptoms, and replacement strategies are considered as potential therapeutics
for neurodegenerative diseases such as Parkinson's, Alzheimer's and Huntington's diseases.
GDNF administration has recently been shown to be an effective treatment for Parkinson's disease, with clin-
ical trials currently in progress. Trials with NGF for Alzheimer's disease are ongoing, with some degree of suc-
cess. Preclinical results using BDNF also show much promise, although there are accompanying difficulties.
Ultimately, the administration of a therapy involving proteins in the brain has inherent problems. Because
of the blood–brain-barrier, the protein must be infused directly, produced by viral constructs, secreted
from implanted protein-secreting cells or actively transported across the brain. An alternative to this is the
use of a small molecule agonist, a modulator or enhancer targeting the associated receptors.
We evaluate these neurotrophic factors as potential short or long-term treatments, weighing up preclinical
and clinical results with the possible effects on the underlying neurodegenerative process.
© 2013 Elsevier Inc. All rights reserved.
Conflict of interest statement
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Glial cell line-derived neurotrophic factor (GDNF)
The neurotrophins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
GDNF, NGF and BDNF in the neurodegenerative disease process . . . . . . . . . . . . . . . . . . . . . .
Parkinson's disease (PD) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Alzheimer's disease (AD), NGF and BDNF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Summary and conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . . . .
Pharmacology & Therapeutics 138 (2013) 155–175
Abbreviations: AAV2, adeno-associated viral vector; AD, Alzheimer's disease; ADAMs, a disintegrin and metalloprotease enzyme family; ALS, Amyotrophic lateral sclerosis; AICD,
APP intracellular domain; apoE, apolipoprotein E; APP, amyloid precursor protein; BACE, beta-site APP-cleaving enzyme; BDNF, brain derived neurotrophic factor; CED,
Convection-enhanced delivery; CEI, (acetyl) cholinesterase inhibitor; ChAT, choline acetyltransferase; ChBF, cholinergic basal forebrain; CNS, central nervous system; CPE, carboxy-
peptidase E; CSF, cerebrospinal fluid; DBS, Deep brain stimulation; DOPAC, dihydroxyphenylacetic acid; DRG, dorsal root ganglia; FDG, fluorodeoxyglucose; GAB1, GRB2-associated
binding protein 1; GFAP, glial fibrillary acidic protein; GDNF, glial derived neurotrophic factor; GFRα, GDNF family receptor α; GPe, globus pallidus external; GPi, globus pallidus
internal; GSK3β, glycogen synthase kinase 3; GPI, glycosylphosphatidyl inositol; GRB2, growth factor receptor-bound protein 2; HAP1, huntingtin-associated protein-1; HD,
Huntington's disease; HVA, homovanillic acid; 6-OHDA, 6-hydroxydopamine; ICV, intracerebroventricular; L-dopa, levo-dopa; LTD, long term depression; LTP, long-term potenti-
ation; MECP2, methyl-CpG binding protein 2; MMSE, Mini-mental status examination scores; MPP, 1-methyl-4-phenylpyridinium; MPTP, 1-methyl-4-phenyl-1,2,3,6-
tetrahydropyridine; NCAM, neural cell adhesion molecule; NFκβ, (nuclear factor κβ; NGF, nerve growth factor; NRSF, neurone-restrictive silencing factor; NSAIDs, non-steroidal
anti inflammatory drugs; NT3, neurotrophin 3; NT4, neurotrophin 4; PCA, p-chloroamphetamine; p75NTR, 75 kDa pan neurotrophin receptor; PD, Parkinson's disease; PET, positron
emission tomography; PI3K, phosphoinositide 3-kinase; PLCγ, phospholipase Cγ; PNS, peripheral nervous system; SH-2, src-homology-2; SNpc, pars compacta of the substantia
nigra; SOD, superoxide dismutase; TBI, traumatic brain injury; TGF-β, transforming growth factor-β; TNF, tumour necrosis factor; tPA, tissue plasminogen activator; Traf, TNF re-
ceptor associated factor; TrkA receptor, Trypomysin receptor kinase A; TrkB, tyrosine kinase receptor B; TrkB-FL, TrkB full-length, catalytic form; TrkB-T, TrkB truncated; UPDRS,
Unified Parkinson's Disease Rating Scale; Vps10p, vacuolar protein sorting 10 protein.
⁎ Corresponding author at: Dorothy Hodgkin Building, University of Bristol, Whitson Street, Bristol BS1 3NY, UK. Tel.: +44 117 3313053.
E-mail address: Shelley.email@example.com (S.J. Allen).
0163-7258/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
Contents lists available at SciVerse ScienceDirect
Pharmacology & Therapeutics
journal homepage: www.elsevier.com/locate/pharmthera
Therapies to replenish endogenous neurotrophic factors by supply-
ing GDNF, BDNF, NGF or suitable mimetics to an appropriate site of ac-
tion, may provide important neuronal support in two of the most
common neurodegenerative diseases, Alzheimer's (AD) and Parkinson's
(PD). This approach may also prove effective for the less common dis-
eases such as Huntington's (HD), amyotrophic lateral sclerosis (ALS),
and Rett Syndrome. Despite success in a multitude of preclinical studies
and early clinical trials, neurotrophic factors have yet to fulfil their po-
tential as neurorestorative therapies. Here we attempt to present these
trophic factors in the context of neurodegeneration and illustrate how
supplementation may complement existing or emerging therapies.
2. Glial cell line-derived neurotrophic factor (GDNF)
GDNF was originally isolated from the supernatant of a rat glioma
cell-line, and found to have pronounced effects on the survival of
midbrain dopaminergic neurons (Lin et al., 1993). It has a relatively
high specificity for dopaminergic neurons and thus has significant po-
tential for the treatment of PD, which is predominantly characterised
by progressive depletion of midbrain dopaminergic cell populations.
Subsequently, GDNF has also been found to have trophic and protec-
tive effects on noradrenergic neurons in the locus coeruleus, as well
as peripheral motor neurons, raising hopes for its therapeutic poten-
tial in HD and ALS (Zurn et al., 1994, 1996; Arenas et al., 1995). Trans-
lational research has focused mainly on the treatment of PD patients,
where there has been cause for both celebration and caution.
2.1. The GDNF superfamily
GDNF and the GDNF-family ligands artemin (Fig. 1), neurturin and
persephin belong to the transforming growth factor-β (TGF-β) super-
cytokines, including the neurotrophin family. Although GDNF shows
only limited amino-acid sequence homology with the other members
of the superfamily, it has significant conformational similarity as they
tion of three disulphide bonds. This family of proteins all function as
homodimers and show protective and restorative effects in the devel-
oping and adult central nervous system (CNS) (Airaksinen & Saarma,
2002). The neurotrophic effects of GDNF appear to be dependent on
the presence of TGF-β in both in vivo and in vitro studies. This syner-
gism is reported to result from TGF-β-induced recruitment of GDNF re-
ceptors to the plasma membrane rather than up-regulation of receptor
expression (Peterziel et al., 2002).
mature protein of 134 amino-acids. Activated GDNF is a disulphide-
bonded homodimer of 32–42 kDa. The first 37 N-terminal amino acids
constitute a high-affinity, heparin-binding domain which interact with
extracellular heparan sulphate glycosaminoglycans. GDNF undergoes
N-terminal cleavage both in vitro and in vivo. Despite lacking the
heparin-binding domain, the truncated form, des37-GDNF, is reported
to retain its full biologic activity and ability to promote dopaminergic
neuronal survival (Xu et al., 1998).
Analyses of tissue distribution of GDNF mRNA shows that it is
expressed in the striatum and skeletal muscle, the target fields for do-
paminergic substantia nigra neurons and motor neurons respectively,
suggesting that GDNF acts as a target-derived neurotrophic factor for
these neurons. In situ hybridization and reverse transcription, poly-
merase chain reaction (PCR) studies have revealed GDNF mRNA ex-
pression in many regions of the developing and adult brain, as well
as in peripheral tissues (including the kidney and testes), indicating
that GDNF has also a role in the regulation of kidney morphogenesis
and spermatogenesis (Yamamoto et al., 1996). GDNF is a potent pro-
moter of neuronal survival in the CNS and peripheral nervous system
(PNS), and has been shown to have effects on a number of cell
populations including sensory and autonomic ganglia, Purkinje cells
of the cerebellum, hippocampal neurons, as well as noradrenergic,
serotoninergic, and cholinergic neurons.
2.2. GDNF receptors
GDNF and related GDNF-family ligands share the Ret protein as a
common signalling receptor. Ret is a tyrosine kinase and the product of
the c-ret proto-oncogene. In keeping with theobserved neuroprotective
ly expressed in the substantia nigra of adult rats (Durbec et al., 1996;
Trupp et al., 1996, 1997). However, for Ret receptors to function in
vivo, they require association with a glycosylphosphatidyl inositol
(GPI)-anchored protein within a multicomponent receptor complex
termed the GDNF family receptor α (GFRα) (Naveilhan et al., 1998).
Four structurally-related GFRα receptors have been identified —
GFRα1, GFRα2, GFRα3 and GFRα4, which are involved in binding of
form a high-affinity complex with GFRα1, resulting in aggregation of
two Ret molecules (Fig. 2). Transphosphorylation of tyrosine residues
signalling cascades including the MAP kinase and phosphoinositide
3-kinase (PI3K) pathways, which are thought to have important roles
in neurite outgrowth and neuronal survival (Chen et al., 2001a,
2001b). GDNF signalling also utilises Src-family kinases, which have
roles in promoting neuronal survival and neurite outgrowth via
PI3K-dependent pathways (Encinas et al., 2001).
In addition to GFRα1 and Ret, effective GDNF signalling requires
the presence of heparin sulphate glycosaminoglycans which facilitate
Ret phosphorylation and axonogenesis (although very high concen-
trations of GDNF can activate Ret in the absence of heparin sulphate)
(Barnett et al., 2002). Interestingly, GDNF can trigger intracellular sig-
nalling cascades independent of Ret via Src-family kinases. In the
forebrain, cortex and inner ear, GFRα receptors are found without
co-expression of Ret suggesting that other transmembrane proteins
might mediate GDNF activity (Trupp et al., 1997). Neural cell adhe-
sion molecule (NCAM) has been identified as a second signalling re-
ceptor for GDNF-family ligands (Paratcha et al., 2003). Binding of
GDNF to NCAM results in Schwann cell migration and axonal growth
Fig. 1. TheX-raycrystalstructuresofGDNFandarteminwithreceptorsGFRα1andGFRα3
respectively. (a) GDNF dimer is in cyan and blue. GFRα1 domains are in magenta. Taken
from 2V5E.pdb which has monomer GDNF and single GFR domain in asymmetric unit.
Dimer generated from symmetry atoms as biologically relevant molecule. (b) Artemin
shown in blue and purple GFRα 3 domains in green. Taken from 2GHO.pdb.
S.J. Allen et al. / Pharmacology & Therapeutics 138 (2013) 155–175
in the hippcampus and cortex — effects which occur independently of
3. The neurotrophins
3.1. Nerve growth factor (NGF)
Nerve growth factor (NGF) (Fig. 3) is a secreted growth factor, im-
portant in survival, growth and maintenance of specific types of neu-
rons in the central and peripheral nervous system. It was first
investigated in a series of experiments, begun over sixty years ago,
when a soluble growth factor, released from sarcoma tissue, was
found to be able to cause outgrowth of fibres from sensory or sympa-
thetic nerve cells placed nearby (Levi-Montalcini & Hamburger, 1951;
Cohen et al., 1954; Levi-Montalcini & Cohen, 1960).
NGF is synthesised as a precursor, proNGF, which is cleaved to
produce the mature form, a non-covalent linked homodimer of ap-
proximately 26 kDa. Like GDNF, NGF is a member of the cysteine
knot superfamily, with the characteristic loop formation with three
disulphide bonds. NGF was the first of the neurotrophin family of
nerve growth promoting factors to be discovered. The “neurotrophic
factor hypothesis” (Yuen et al., 1998), described subsequent to the
discovery of NGF, proposed that developing neurons die if they fail
to compete for limited supplies of a target-derived neurotrophic fac-
tor. In accordance with this, NGF has been shown to be produced in
target tissues of sensory and sympathetic fibres, taken up by the
fibre terminals, and retrogradely transported back to the nerve cell
body where it is required for the survival and maintenance of these
neurons. NGF mRNA levels are notably high in the spinal cord and
dorsal root ganglia (DRG) (Sobue et al., 1998) and it is crucial during
Fig. 2. A simplified schematic of some of the signalling pathways via Ret. GDNF dimer binds to two GFRα1 receptors and dimerises two RET receptors on the surface of the neurone
enabling signalling to the nucleus. The major pathways involved are via (i) Ras/ERK1/2; (ii) PLCγ (phospholipase Cγ) and (iii) PI3K (phosphoinositide 3-kinase)/Akt (protein kinase
B). In a similar way, neurturin is able to bind GFRα2, artemin binds GFRα3 and persephin binds GFRα4, which then, in turn, bind to RET receptors. Crystal structures are given
where known. GFRα is a glycophosphotidylinositol (GPI) anchored receptor.
S.J. Allen et al. / Pharmacology & Therapeutics 138 (2013) 155–175
development for the survival and maintenance of trigeminal dorsal
root sensory neurones and superior cervical ganglion. Thus, mice
with homozygous knockout of NGF have severe sympathetic and sen-
sory deficits and are not viable for more than a few weeks after birth
(Crowley et al., 1994; Huang & Reichardt, 2001). This is also true for
mice with homozygous knockout of the NGF tyrosine kinase receptor
TrkA (Tropomysin Receptor Kinase A; the name reflects its original dis-
covery (Martin-Zanca et al., 1986). See Section 3.3 and Fig. 3 for more
detail of the receptors) (Smeyneet al., 1994; Huang & Reichardt, 2001).
In the brain, NGF is produced in the cortex and hippocampus,
which are targets of projection neurons from the cholinergic basal
forebrain (ChBF) and some striatal and hippocampal neurones. Inter-
estingly, in contrast to the severe sympathetic and sensory neurone
deficits, homozygous NGF knockouts, perinatally and during their
few weeks of survival, have an intact cholinergic basal forebrain
(Crowley et al., 1994). However, by contrast TrkA null mice show a
substantial decrease in cholinergic basal forebrain hippocampal and
cortical projections (Smeyne et al., 1994). Further analysis of these
mice shows that in the first week postnatal, cholinergic neurones
are present but smaller than normal; by four weeks there are signifi-
cant depletions in neuronal number and cholinergic innervation of
the hippocampus is abnormal (Fagan et al., 1997). Furthermore,
mice, heterozygous for NGF knockout, survive to adulthood and can
be tested for cognitive impairment. These have a substantial reduc-
tion in cholinergic innervation of the hippocampus and resultant
memory deficits (Chen et al., 1997), and suggest that NGF is required
throughout maturity for cholinergic–hippocampal interaction.
tors are brain derived neurotrophic factor (BDNF), neurotrophin 3
(NT3) and neurotrophin 4 (NT4) (Fig. 3). These are all survival factors,
essential for the appropriate development and subsequent survival
and maintenance of specific subsets of neurons into adulthood. In
1982 BDNF, was purified from pig brain (Barde et al., 1982). Following
the cloning of NGF (Scott et al., 1983) and later, BDNF (Leibrock et al.,
1989), PCR cloning was employed to search for, and discover, NT-3
(Maisonpierre et al., 1990), Xenopus NT-4 (Hallböök et al., 1991) and
subsequently the human homologue of NT-4 (Berkemeier et al., 1991).
These neurotrophins, like NGF, are all produced aspro-forms, which
are cleaved either intra- or extra-cellularly to form the mature protein.
Theyexist biologicallyashomodimers andthecrystalstructuresofeach
of the mature proteins have been solved (Fig. 3). The neurotrophin
pro-region for a long time was viewed simply as a requirement for cor-
rect folding ofthe mature protein.Now the pro-neurotrophin forms are
seen to have specific functions, often in direct opposition to the roles of
their mature counterparts (Lu et al., 2005).
3.2. Brain derived neurotrophic factor (BDNF)
BDNF supports the survival and maintenance of sensory neurons,
retinal ganglia, certain cholinergic neurons, spinal motor neurons
and some dopaminergic neurons. In the brain, the synthesis of
BDNF, as investigated in the hippocampus and cortex, is affected by
neuronal activity and has a unique role in synaptic plasticity. The
use or disuse of synaptic pathways results in strengthening or
Fig. 3. X-ray crystal structures of the neurotrophins and their receptors (a) The X-ray crystal structures of the neurotrophins. NGF (cyan) is taken from 1WWW, the BDNF structure
is a modelled homodimer using 1BND.pdb template. NT3 (orange) is from 1BUK.pdb in complex with two p75 receptors. NT4 (blue) is taken from 1HCF.pdb, in complex with TrkB.
(b) Structures of receptors: TrkA (pink)–NGF (cyan) (2IFG), p75NTR (green) binding NT3 (orange) (3BUK), sortilin (blue) is taken from 3F6K.pdb in complex with neurotensin.
Apart from TrkA, only the d5 (Ig2) domain of each of the Trk receptors has been solved. However it is assumed that the full 3D structure of the other Trk extracellular domains
will be similar to TrkA. The extracellular region of the Trk receptors can be sub-divided (by amino acid sequence) into different domains (d1–d3 is a leucine-rich, cysteine-rich
(LR&CR) region). Domains 4 and 5 are immunoglobulin-like domains. d5, the domain closest to the membrane, binds the mature neurotrophin directly. Full length p75NTR has
four cysteine-rich repeats in the extracellular domain. Dotted lines represent unknown protein conformation between the crystal structure and the plasma membrane (~40 residues
for TrkA, ~60 residues for p75NTR).
S.J. Allen et al. / Pharmacology & Therapeutics 138 (2013) 155–175
weakening of connections between neurons, resulting in increased or
decreased formation of synapses at axon collaterals and dendritic
spines. A major element of the strengthening of synaptic connections
involves long-term potentiation (LTP). BDNF has a pivotal role in this,
facilitating the synthesis and consolidation of new memories.
The BDNF gene is one of the most tightly regulated genes, with 11
exons and 9 functional promoters (Pruunsild et al, 2007) and gives
rise to approximately 34 transcripts (Gabriele & Tongiorgi, 2009) in re-
are released via the constitutive pathway, BDNF can also be secreted via
an activity-dependent regulated secretory pathway in neuronal cells.
Processing by proBDNF or proNGF to the mature form may occur inside
or outsidethecell. Intracellularprocessing is via furin;extracellularpro-
cessing occurs via plasmin, generated following the activation of plas-
minogen by tissue plasminogen activator (tPA), which is co-released
with proBDNF (Pang et al., 2004). During regulated secretion, BDNF
(the mature domain) associates with a sorting receptor carboxypepti-
dase E (CPE), whilst the pro domain binds the sortilin receptor
ectodomain (Zhang et al., 1999). ProBDNF also associates with the
huntingtin-associated protein-1 (HAP1); sortilin stabilises the complex
sists intheintracellular cleavage ofproBDNFby furintoproducemature
BDNF, and release by the secretory pathway (Yang et al., 2011). A com-
mon polymorphism (proBDNFVal66Met) precludes the binding of
proBDNF to sortilin which is thus not able to undergo regulated secre-
tion. There are a number of consequences to this, one of which may be
impairment of memory function (Egan et al., 2003).
BDNF and the activation of its tyrosine kinase receptor TrkB is im-
portant in late phase LTP (L-LTP), which requires protein synthesis
and promotes structural changes at the synapse. In heterozygous
BDNF knockout mice, or if BDNF is removed by antisense, L-LTP is di-
minished (Korte et al., 1995) but can be rescued by, for instance, viral
transfer of BDNF (Korte et al., 1996).
facilitates hippocampal LTD by increasing the NMDA receptor NR2B
subunit (Woo et al., 2005). p75NTR (75 kDa pan neurotrophin recep-
tor) is present in CAI neuron dendritic spines in mouse hippocampus;
and in its absence there is a decreased expression of NR2B and impair-
ment of proBDNF-initiated LTD (Woo et al., 2005). Since loss of synap-
ses is an important early feature of AD, the role of LTD in AD is likely
to be an important factor (Koffie et al., 2011).
3.3. Neurotrophin receptors
The mature neurotrophins each bind with picomolar affinity to
their specific tyrosine kinase receptor. NGF binds TrkA, BDNF and
NT4 bind TrkB, and NT3 binds TrkC. For each Trk receptor, only the
domain closest to the membrane (called d5 or Ig2) is required to
bind to its neurotrophin ligand (Wiesmann et al., 1999; Banfield et
al., 2001; Naylor et al., 2002). All the neurotrophins bind to p75NTR
in the low nanomolar affinity range (Chao, 1994). Fig. 3 shows the
elucidated structures of the human neurotrophins and the receptors
TrkA, p75NTR and sortilin.
Binding of the neurotrophins to their Trk receptors initiates
dimerisation of two receptors and trans-phosphorylation of certain
tyrosine residues within the intracellular domains. Adapter proteins
which have src-homology-2 (SH-2) or phosphotyrosine-binding mo-
tifs are then recruited to these sites of phosphorylation. These initiate
intracellular signalling cascades (Fig. 4) which may lead to neuronal
differentiation and/or survival and gene expression.
The presence of p75NTR assists binding of (and discrimination for)
the major neurotrophin ligand at its Trk receptor (e.g. NGF for TrkA)
(Chao & Hempstead, 1995). However, importantly, in the absence of
the Trk receptors or the neurotrophins, p75NTR may initiate mecha-
nisms leading to apoptosis (Blochl & Blochl, 2007). p75NTR is a
member of the tumour necrosis factor (TNF) receptor superfamily
(Gruss & Dower, 1995). Its extracellular domain has four negatively
charged cysteine repeats, the third and the fourth of which form the
ligand binding region. The intracellular region comprises a ‘chopper’
domain and a ‘death’ domain which promote apoptosis (Coulson et
al., 2004). It is able to bind the pro- and mature forms of the
neurotrophins, and interacts with several other proteins including
NRAGE, NRH2 and NRIF to promote neurite outgrowth, proliferation
and cell death (Chao et al., 2006).
p75NTR in the adult forebrain, is mainly located on neurons in the
ChBF and its projections, with some present in certain parts of the
hippocampus (Allen et al., 1989; Sobreviela et al., 1994). TrkA is
also expressed in these neurons, and additionally in the striatum
and hippocampus (Steininger et al., 1993). TrkB and TrkC are also
present on the ChBF cells but are also much more widely distributed
(Altar et al., 1994). In human brain, there are three main isoforms of
TrkB: a full-length, catalytic form (TrkB-FL), and two ‘truncated’
forms which lack the tyrosine kinase domain: truncated (TrkB-T)
and TrkB-Shc. TrkB-FL is able to initiate survival, differentiation and
synaptic plasticity signals, and it is proposed that the truncated
forms act to inhibit these effects by heterodimerisation with TrkB-FL
(Wong et al., 2011).
More recently, sortilin has been shown to be a receptor for the
pro-neurotrophins (Nykjaer et al., 2004), but not the mature
neurotrophins. It is a member of the vacuolar protein sorting 10 pro-
tein (Vps10p) domain containing family, produced as a proform and
processed to the mature form mainly by furin in the trans-Golgi net-
work. In addition to the pro-neurotrophins, mature sortilin has a
number of binding partners including neurotensin and progranulin
(Nykjaer & Willnow, 2012).
This is only a brief summary of the neurotrophins and their cellular
effects. The signalling of the neurotrophins via their receptors initiates
various signalling pathways, which can be modified by a number of
criteria with cell dependant outcomes. The diversity of their effects
can be seen by the fact that NGF and BDNF are important in pain me-
diation in the periphery, and also in cholinergic–hippocampal com-
munication and memory formation in the brain. The following
sections present examples of how deficiency of neurotrophins or
GDNF may have detrimental effects on brain function and how
their restoration or supplementation may slow or reverse these
4. GDNF, NGF and BDNF in the neurodegenerative disease process
In 1981 a hypothesis was presented by Stanley Appel (Appel,
1981) entitled ‘A unifying hypothesis for the cause of amyotrophic
lateral sclerosis, parkinsonism, and Alzheimer disease’. This suggested
that each of these disorders was due to the lack of a ‘hormone’ or
growth factor, which would normally be secreted by the target tissue
of affected neurons and retrogradely transported after being taken up
by the presynaptic terminal. In amyotrophic lateral sclerosis (ALS)
muscle cells would fail to release the appropriate factor resulting in
loss of function of anterior horn cells, in PD striatal cells would fail
to produce a dopaminergic factor, resulting in loss of substantia
nigra cells and in AD the hippocampal and cortical cells would fail
to supply the cholinergic factor with resultant loss of function of the
Although perhaps never quite viewed as a solution to the causa-
tion of each of these diseases there is still much to recommend this
concept. BDNF and GDNF are both known to be important for dopa-
minergic and motor neuron survival, NGF and BDNF for cholinergic
neurons, and BDNF is also important in the survival and function of
serotonergic, hippocampal and cortical neurons. Reduction in BDNF
levels have been associated with a number of neurodegenerative, de-
velopmental and neuropsychiatric conditions including AD (Phillips
et al., 1991; Connor et al., 1997; Ferrer et al., 1999; Hock et al.,
S.J. Allen et al. / Pharmacology & Therapeutics 138 (2013) 155–175
2000; Holsinger et al., 2000; Peng et al., 2005), PD (Howells et al.,
2000), HD (Zuccato et al., 2008), ALS (Nishio et al., 1998), Rett syn-
drome (Sun & Wu, 2006), schizophrenia (Favalli et al., 2012) and de-
pression (Castren et al., 2007).
Deficits of these growth factors, even if not the initial trigger for
the disease process, may lead to increased cell death with resulting
symptoms. Some examples of this are outlined briefly below. This re-
view deals mainly with neurodegeneration, with a mention of Rett
syndrome, other reviews deal in more detail in particular with
BDNF and neuropsychiatric disorders (Autry & Monteggia, 2012;
Balaratnasingam & Janca, 2012).
4.1. Huntington's disease (HD)
HD is an autosomal dominant neurodegenerative disorder in
which the protein huntingtin has an N-terminal polyglutamine ex-
pansion, resulting from CAG trinucleotide repeats in the huntingtin
(htt) gene. It is a progressive choreic movement disorder, often
with cognitive dysfunction and emotional disturbance. The neuro-
pathological changes include cell loss, particularly in the neocortex,
and striatum with a selective degeneration of medium sized spiny
projection neurones (Graveland et al., 1985); resulting in the motor
dysfunction characteristic of the disease. These striatal neurones re-
quire BDNF, provided by the cortical neurones, for survival and differ-
entiation, and it has been suggested for some time that the loss of
BDNF may account for much of the symptomatology of this disease.
In fact BDNF cortical tissue levels may be reduced by up to 45% in
HD (Ferrer et al., 2000; Zuccato et al., 2001); and support for the
striatal neurones is worsened by impaired huntingtin-mediated
BDNF transport from the cortex (Gauthier et al., 2004).
Transgenic animal models of HD with increased CAG repeats, show
similar changes and also BDNF function and transport are similarly af-
fected. Whereas overexpression of wild type huntingtin protein in cell
lines and in ‘Htt knock in’ transgenic mice, results in increased levels
in BDNF production (Reilly, 2001; Canals et al., 2004; Zuccato &
Cattaneo, 2009). BDNF is thus reduced in striatum and cortex of HD
transgenic mice overexpressing the mutant huntingtin.
The level of BDNF affects severity of HD symptoms, as shown in
HD transgenic mice, which have also been crossed such as to be het-
erozygous for BDNF knockout; these have a higher degree of severity of
symptoms. The mechanism by which BDNF is downregulated is at least
partly due to the increased binding of the neurone-restrictive silencing
factor (NRSF) to the BDNF promoter (Zuccato et al., 2003), thus
Fig. 4. A simplified schematic of some of the signalling pathways via Trk or p75NTR. 1) Three main pathways of Trk involve phosphorylation of (i) shc/GRB2 (growth factor
receptor-bound protein 2)/GAB1 (GRB2-associated binding protein 1) and Ras/ERK1/2; (ii) PLCγ/PIP2 (diphosphobisphosphate)/PKC and (iii) PI3K (phosphoinositide 3-kinase)/
Akt (protein kinase B). 2) The p75NTR receptor signals via Trafs (TNF receptor associated factors)/ceramide and NFκβ (nuclear factor κβ).
S.J. Allen et al. / Pharmacology & Therapeutics 138 (2013) 155–175
suppressing BDNF transcription. Wild-type huntingtin sequesters NRSF
in the cytoplasm and inhibits its passage into the nucleus and thus pre-
vents its silencing activity; transcription of BDNF is therefore increased.
accumulate in the nucleus and inhibit transcription of certain proteins
A second mechanism has been elucidated to explain the reduction
in BDNF seen in HD. Huntingtin protein is important in regulation of
vesicle transport; it carries out this function at least partly in conjunc-
tion with dynein and the huntingtin associated proteins HAP1 and
HAP40. By contrast, mutant huntingtin is unable to fulfil this function
which results in a reduction in BDNF trafficking (Zuccato & Cattaneo,
BDNF has been shown as having a degree of success in ameliorat-
ing symptoms in a number of studies in preclinical models of HD
(Perez-Navarro et al., 2000; Lynch et al., 2007; Gharami et al., 2008;
Giralt et al., 2009). However, delivery of growth factor proteins into
the brain provides difficulty due to their inability to cross the
blood–brain-barrier (BBB) or cerebrospinal fluid (CSF)–brain barrier.
This makes the form of therapy intrusive by necessity. Also, because
of the nature of neurodegenerative illnesses, these proteins need to
be given chronically.
In accord with circumventing these problems, drugs which in-
crease BDNF activity, such as serotonin-selective reuptake inhibitors
and ampakines, have been studied for their effects in HD models
and have been shown to have beneficial effects (Duan et al., 2008;
Simmons et al., 2009). However, an interesting study published re-
cently presents an alternative way to overcome this problem (Giralt
et al., 2010, 2011). Giralt and colleagues produced a transgenic HD
mouse model cross which also expressed BDNF under control of the
glial fibrillary acidic protein promoter (pGFAP). Striatal parenchymal
samples from both HD patients and HD mouse models have been
shown to have increased GFAP levels and astrogliosis. Therefore,
due to this physiological/pathological upregulation of GFAP, there
was also a concomitant upregulation of striatal BDNF production in
the pGFAP-BDNF HD mouse model. The study went on to demon-
strate that this recovery of BDNF levels correlated with improved
motor coordination and reduced anxiety in the mouse. There was
also an indication of improved cortico-striatal connectivity.
Given the considerable problems which are associated with the
symptoms of HD, for both patients and their carers, preclinical studies
such as these described here provide some cause for optimism, albeit
cautious, for the therapeutic potential of BDNF in HD.
4.2. Amyotrophic lateral sclerosis (ALS)
ALS (also called Lou Gehrig's disease) is a disease primarily involv-
ing the death of the motor neurones in the spinal cord, brain stem and
motor cortex (Kiernan et al., 2011). It is described as a system degen-
eration disorder in which there is progressive degeneration of both
upper motor corticospinal and lower motor spinal neuronal tracts.
These tracts control numerous motor functions including breathing
and swallowing, which can be profoundly affected in ALS with fatal
consequences. This disease is currently incurable.
With an onset typically in the fifties, limbs gradually weaken, with
death often occurring within three years of diagnosis, usually due to
respiratory problems. A minority of sufferers have dementia, thought
to be due to frontal lobe degeneration.
ALS symptoms are accompanied by a neuropathology which in-
cludes neuronal loss, astrocytosis and deposition of abnormal phos-
phorylated neurofilament in the neuronal cytoplasm. One of the early
models of spinal muscular atrophy was the wobbler mouse (Falconer,
1956),whichoriginated from a spontaneousmutation.This mouse pre-
sents at an early age with a wobbly gait, associated with motoneuronal
degeneration in the spinal cord and the brainstem.
gene encoding Cu/Zn superoxide dismutase, a free-radical-scavenging
enzyme. More than a hundred different mutations in SOD1 have been
linked to familial ALS, the vast majority of which are mutations which
change one amino acid.
The familial and sporadic forms of ALS have similar symptoms and
neuropathology (Bendotti & Carrì, 2004); transgenic mice expressing
mutant SOD1 also develop motor neurone-like disease and act as use-
ful models (Gurney et al., 1994). Hypotheses as to the cause of the pa-
thology in sporadic ALS include increased reactive oxygen species
(Carter et al., 2009) and glutamate excitotoxicity (Corona et al.,
2007). However, since SOD1 knockout mice are more or less symp-
tom free, it is thought that these mutations must be associated with
a gain of toxic function.
GDNF was found to rescue motor neurones after axotomy-induced
degeneration (Buj-Bello et al., 1995; Gimenez et al., 1997) and a num-
ber of studies show motor neurones of SOD1 mice are protected by
this factor (Mohajeri et al., 1999; Li et al., 2007; Park et al., 2009).
However, in vivo the beneficial effects were less consistent (Mohajeri
et al., 1999; Suzuki et al., 2007; Park et al., 2009). Interestingly GDNF
ever, GDNF has not yet been tested in the clinic; if and when it is tested
then, as it does not cross the BBB, delivery would be a problem.
for motor neurones. It has been shown to have beneficial effects on
axotomized motor neurones (Sendtner et al., 1992; Gimenez et al.,
1997) and in wobbler mice (Ikeda et al., 1995). SOD1 transgenic mice
given human neural progenitor cells engineered to express BDNF did
not improve symptoms or survival (Park et al., 2009) although GDNF-
or IGF-1-expressing progenitor cells did reduce the loss of motor
lung function (The BDNF Study Group, 1999). However, this beneficial
effect was not repeated in a larger trial of over a thousand patients
given up to 100 μg/kg recombinant methionyl BDNF (rhmetBDNF); no
improvement was seen (The BDNF Study Group: Phase III, 1999). In a
later double-blind, sequential, dose-escalation study of 25 ALS patients,
BDNF was infused intrathecally for 3 months (up to 1 mg/day). Detect-
able BDNF levels were seen in the CSF, particularly at the lumbar level.
Little improvement was reported but no conclusion could be drawn
about efficacy due to the low patient number (Ochs et al., 2000). The
same was true for later trials, again conducted with small patient num-
bers. ALS patients (n=11) in 2003 received BDNF intrathecally, and in
2005 (n=13) (Kalra et al., 2003; Beck et al., 2005).
ALS however. There are problems associated with intrathecal BDNF pro-
tein delivery, in particular passage across the CSF–brain barrier, and the
short half life and low distribution rate of the protein, both of which
may have contributed to the disappointing clinical results. The presence
of large numbers of truncated TrkB receptors in the brain can mop up
ally, BDNF, like the other neurotrophins, binds to the receptor p75NTR,
which has been described as being implicated in ALS neuropathology
(Dupuis et al., 2008). In fact an antagonist for p75NTR has been shown
to slow ALS progression in SOD1 mice (Turner et al., 2004).
Again, obstacles still need to be overcome to give BDNF-associated
treatment a fair trial as a potential therapy for this extremely debili-
4.3. Rett syndrome
imately 1 in 10,000, mainly females, causing impaired growth and
S.J. Allen et al. / Pharmacology & Therapeutics 138 (2013) 155–175
cognitive development, respiratory problems, gastrointestinal disor-
ders, seizures and autism-spectrum type behaviour (Rett, 1966;
Hagberg et al., 1983). This disorder is caused by mutations in the
methyl-CpG binding protein 2 gene, MECP2 (Amir et al., 1999).
MECP2 binds to a BDNF promoter and is able to regulate BDNF expres-
sion. Further information is given on the likely mechanisms involved in
other reviews (Matsuishi et al., 2011; Autry & Monteggia, 2012).
NGF levels seem to be decreased in the CSF of Rett patients, with
BDNF unchanged compared to normal (Lappalainen et al., 1996;
Riikonen & Vanhala, 1999; Riikonen et al., 2003). In the MECP2 mu-
tant mouse model, as with Rett patients, there is a breathing dysfunc-
tion and reduced BDNF expression and TrkB activation in the medulla
and pons. Disease progression in the MECP2 mice increased or de-
creased as BDNF was conditionally deleted or overexpressed. BDNF
overexpression increased lifespan and rescued locomotor activity
(Chang et al., 2006).
BDNF-associated therapy has been suggested (Sun & Wu, 2006).
As discussed above for HD and ALS patients, of great importance
would be a method of delivery to ensure that the protein reached
the target areas. With this in mind, descriptions are given
(Section 5) surrounding the delivery of GDNF to patients with PD.
This provides insight into why many clinical trials involving delivery
of proteins to the brain show lack of efficacy in the clinic.
Described below in more detail are two of the more common con-
ditions, PD and AD, which may currently be amenable to GDNF, BDNF
and NGF as options for recovery.
5. Parkinson's disease (PD)
PD is a progressive neurodegenerative disorder which affects over
120,000 people in the UK and approximately 1,000,000 in the US. The
clinical course is characterised by impairment of motor function due
to the progressive death of selected populations of dopaminergic
neurones, particularly within the pars compacta of the substantia
nigra (SNpc). There are approximately 500,000 specialised dopami-
nergic cells in the SNpc of young adults which innervate the caudate
nucleus and putamen forming the nigrostriatal dopaminergic path-
way. While the loss of striatal dopamine correlates with the severity
of clinical disability, motor deficits are not apparent until 80–85% of
SNpc neurones have degenerated and striatal dopamine levels are de-
pleted by 60–80% (Patel & Gill, 2007).
The classical features of PD are resting tremor, muscle rigidity,
bradykinesia and loss of postural reflexes. Accompanying signs in-
clude a mask-like facial expression, flexed posture, festinating gait,
handwriting changes, cognitive decline and postural deformities
(Doherty et al., 2011). Signs of parkinsonism are also common in
AD and the cognitive decline in AD is found to be associated with
the progression of parkinsonism. Neurodegenerative changes in PD
are not restricted to dopaminergic neurones and also affect choliner-
gic, noradrenergic and serotoninergic cell populations resulting in a
wide range of clinical manifestations.
The causes of PD are unknown but considerable evidence sug-
gests a multifactorial aetiology involving genetic and environmen-
tal factors. Mutations in the gene encoding α-synuclein have been
identified in some families with autosomal dominant PD. In addi-
tion, deletions in the parkin gene, a gene encoding a ubiquitin
ligase, have been identified in autosomal recessive juvenile forms
of the disease (Bonifati, 2005). Other possible causes of selective
degeneration of nigral dopaminergic neurones in PD include de-
fects in mitochondrial metabolism, mitochondrial gene deletions,
constitutive metabolic deficiencies and cellular damage due to oxi-
dative injury. Fig. 5 attempts to give a brief overview of the molec-
ular pathological processes known in PD and possible therapeutic
Early treatment of the disease was neurosurgical, with a number
of approaches proving beneficial. Thalamotomy was found to relieve
tremor and rigidity, but not bradykinesia; and lesioning of the
globus pallidus internus (pallidotomy) and the subthalamic region
(subthalamotomy) were found to significantly improve all three of the
classical features of PD. Invasive treatments were largely superseded by
the introduction of orally administered L-3/4-dihydoxyphenylalanine
(L-dopa) in the late 1960s. L-dopa is the immediate precursor of dopa-
mine but, unlike dopamine, it is able to effectively cross the BBB.
L-dopa, when combined with an aromatic amino acid decarboxylase in-
hibitor, remains the most effective drug treatment currently available
Whilst helpful in the early stages of PD, treatment with L-dopa and
other dopamine agonists is associated with complications and re-
duced efficacy as the disease progresses. Prolonged use of dopamine
agonists is associated with both motor and psychiatric complications
in a significant proportion of patients. Motor fluctuations can take the
form of distinct “wearing-off” and “on–off” periods. “Wearing-off” or
“end-of-dose deterioration”, describes a relatively gradual and pre-
dictable decline in response to L-dopa that occurs over time, and con-
trasts with “on–off” fluctuations in motor performance that are not
clearly related to L-dopa dosing.
Consequently, PD is an attractive candidate for neurotrophic ther-
apy for a number of reasons. Firstly, current treatments address the
symptoms and not the cause of the disease, and are associated with
a high rate of complications. Secondly, the disease is (predominantly)
characterised by the degeneration of a single localised cell type,
which represent a discrete therapeutic target. The large size of the
GDNF homodimer structure makes it unsuitable for transport across
the BBB, and as a consequence, translational research has focused
on direct delivery of GDNF to the brain.
A number of strategies for delivery of GDNF to the brain have been
investigated in pre-clinical models of PD. In addition to delivery of the
controlled drug release over a prolonged period (Garbayo et al., 2009,
2011). Advances in nanoparticle technology have been applied to
GDNF gene therapy, with promising reports from preclinical studies
using DNA nanoparticle gene transfer to achieve long term GDNF ex-
pression (Fletcher et al., 2011) and to promote the survival of grafted
lated fibroblasts transfected to produce GDNF has also been shown to
confer behavioural improvements in the 6-hydroxydopamine rat
model of Parkinson's disease (Grandoso et al., 2007).
However, the translational potential of implanting microparticles
or transfected cells in the human brain is limited by their size,
which is substantially larger than the effective pore size of the extra-
cellular spaces of the brain. Consequently, the limited distribution of
these vehicles for GDNF delivery is likely to restrict their therapeutic
potential. Whilst advances in nanocarrier technology could facilitate
drug distribution through clinically-relevant brain volumes, ques-
tions surrounding their safety and toxicity for clinical applications
are yet to be answered (Nel et al., in press). In this section we
focus on delivery of the unmodified neurotrophin and viral
vector-mediated gene therapy — strategies which have been previ-
ously approved for clinical trials.
5.1. Preclinical studies of GDNF delivery for PD
The therapeutic effects of GDNF have largely been assessed in
preclinical models utilising experimental lesions of the nigrostriatal
pathway. The commonest forms of lesioning are the 6-OHDA
tetrahydropyridine) models, which demonstrate selective depletion of
nigral dopaminergic neurones. These models offer a rapid and repro-
ducible method of inducing parkinsonian motor symptoms, as well as
quantifiable motor outputs, but also have significant limitations of ap-
plicability to the human disease process (Hirsch, 2007).
S.J. Allen et al. / Pharmacology & Therapeutics 138 (2013) 155–175
5.2. Intraventricular administration of GDNF in rodents
Direct infusion of recombinant human GDNF and125Iodine-labelled
GDNF into the lateral ventricle of normal rats has been shown to result
hypothalamus, substantia nigra/ventral tegmental area and cerebellum
(Martin et al., 1996a; Lapchak et al., 1997a). This strategy has been
found to significantly increase striatal and nigral dopamine levels, but
also to increase hypothalamic dopamine content, which might explain
the reduced food and water consumption and body weight gain ob-
served in experimental animals. Intraventricular infusion of GDNF into
aged and 6-OHDA-lesioned rats has also been shown to result in im-
proved locomotor performance and increased striatal dopamine
turnover (Bowenkamp et al., 1997; Lapchak et al., 1997b, 1997c).
However, reduced weight gain has been reported as a consistent
The small volume of the rodent brain in comparison to the human
brain raises significant questions over the applicability of these posi-
tive findings to clinical trials. Consequently, intraventricular infusion
of GDNF has also been performed in non-human primates.
5.3. Intraventricular administration of GDNF in non-human primates
A number of studies of intraventricular infusion of GDNF in
MPTP-lesioned rhesus monkeys and marmosets have been performed
with mixed results. Significant improvements in locomotor acitivity
in hemiparkinsonian rhesus monkeys were demonstrated following
four monthly infusions at doses ranging from 100 to 1000 μg of
GDNF (Zhang et al., 1997). Improvements in motor function have
been correlated with increases in tissue dopamine and dopamine
metabolite concentrations in the substantia nigra but not the puta-
men (Gerhardt et al., 1999). Promising improvements in motor dis-
ability and reductions in L-dopa-induced dyskinesia have also been
observed in marmosets receiving intraventricular GDNF infusions
(Costa et al., 2001; Iravani et al., 2001).
However, in an autoradiographic study of the distribution of125I-
GDNF infused into the lateral ventricle of MPTP-lesioned rhesus mon-
keys, GDNF did not appear to diffuse readily into the caudate/putamen
(Lapchak et al., 1998). This finding contrasted to comparable rodent
studies, suggesting that the success of intraventricular infusions in ro-
the rat brain. Furthermore, weight loss and dyskinesiae were found to
be recurrent complications in non-human primate studies.
5.4. Localised intraparenchymal delivery of GDNF in rodents
Injection of GDNF into the substantia nigra or striatum of
6-OHDA-lesioned rats has been shown to be both neuroprotective, if
administered at the time of lesioning, and neurorestorative if delivered
after lesioning (Sauer et al., 1995a, 1995b; Kearns et al., 1997; Sullivan
et al., 1998).
5.4.1. Neuroprotective effects of GDNF in rodent studies
Intrastriatal delivery of 6-OHDA induces acute toxicity of striatal ax-
onal terminals followed by retrograde degeneration and cell death
within the substantia nigra. Delivery of GDNF into the substantia nigra
generation, but is very effective at preventing retrograde cell death
within the substantia nigra (Winkler et al., 1996). Nigral cells can sur-
vive for months even following withdrawal of GDNF. However, this
therapeutic strategy does not result in recovery from lesion-induced
Fig. 5. A simplified schematic of some of the pathological processes in PD and potential therapeutic targets. This does not intend to be inclusive of either pathology or therapeutics.
The pathways are given between the areas striatum (caudate and putamen), substantia nigra pars compacta (SNpc), globus pallidus external (GPe) and internal (GPi), subthalamic
nucleus (subth n) thalamus and cholinergic basal forebrain (ChBF). Loss of the dopaminergic signalling leads to reduced dopaminergic striatal tone resulting in an imbalance be-
tween dopamine and acetylcholine pathways. Therapeutic targets: T1: Levo-dopa (L-dopa) or dopamine agonists; T2: growth factors GDNF and BDNF, by protein infusion, AAV de-
livery or by small molecule agonists; GDNF infusion into the postero-dorsal (sensorimotor) putamen is retrogradely transported to the substantia nigra down surviving
dopaminergic neurones leading to upregulation, neuroprotection and neurorestoration through neurite branching T3: Deep brain stimulation — electrodes into thalamus or
more recently stimulation of the subthalamic nucleus for tremor, rigidity, bradykinesia and pedunculopontine nucleus for postural instability. Surgery-pallidotomy to lesion the
overactive globus pallidus to prevent bradykinesia, tremor and rigidity; thalamotomy to lesion part of the thalamus to block tremor. T4: antioxidants to combat oxidative stress.
S.J. Allen et al. / Pharmacology & Therapeutics 138 (2013) 155–175
motor deficits. In contrast, intrastriatal delivery of GDNF at the time of
striatal lesioning protects the entire nigrostriatal dopaminergic path-
way, resulting in preservation of locomotor function and suggests that
the effect of GDNF on dopaminergic neurones differs when applied to
axons or cell bodies (Kirik et al., 2000).
5.4.2. Neurorestorative effects of GDNF in rodent studies
Administration of GDNF following 6-OHDA lesioning results in
both neurochemical and functional changes. When administered
into the substantia nigra, significant increases in nigral (but not
striatal) tyrosine hydroxylase activity are detected, and are associated
with increased release of dopamine and its metabolites (Lapchak et
al., 1997b). Such neurochemical changes are accompanied by im-
provements in spontaneous and amphetamine-induced locomotor
activity (Martin et al., 1996b).
GDNF-induced functional improvements following severe 6-OHDA
lesioning of the striatum appear to be primarily mediated by neuro-
chemical changes in downstream structures from the striatum — most
prominently the globus pallidus and substantia nigra, rather than with-
in the striatum itself (Lapchak et al., 1997c). In rats with only partial
lesioning of the striatum, intrastriatal delivery of GDNF results in tran-
sient improvements in motor function, which diminish on withdrawal
of GDNF suggesting that in less severe cases, motor recovery may be
mediated in part by restoration of striatal terminals (Kirik et al., 2001).
5.4.3. Intrastriatal delivery of GDNF in non-human primate studies
A number of studies have investigated the effects of continuous
intraputamenal delivery of GDNF at doses ranging from 7.5 to
22.5 μg/day in both intact aged and MPTP-lesioned non-human pri-
mates (Maswood et al., 2001; Grondin et al., 2002; Ai et al., 2003;
Grondin et al., 2003). Histological analysis confirmed increases in do-
paminergic cell size and number within the substantia nigra, as well
as increased fibre density in the caudate nucleus, putamen and globus
pallidus. Biochemical analysis demonstrated increases in dopamine
and the dopamine metabolites dihydroxyphenylacetic acid (DOPAC)
and homovanillic acid (HVA) in the striatum and globus pallidus.
MPTP-lesioned primates showed improvements in the primate PD
rating scale whilst intact aged monkeys demonstrated improvement
in general motor performance at high doses and increases in hand
5.4.4. Chronic infusion of GDNF and related toxicity
The results of a six month chronic infusion toxicity study in rhesus
monkeys cast significant doubts over the neurorestorative potential
of GDNF for the treatment of PD (Hovland et al., 2007). In this study
primates received 0, 15, 30 or 100 μg/day recombinant human
GDNF by continuous unilateral intraputamenal infusion at a flow
rate of 150 μl/day. In the highest treatment group, a number of path-
ological markers of toxicity were observed including reduced food in-
take and weight loss, meningeal thickening and most worryingly,
multifocal cerebellar Purkinje cell loss. The authors concluded that
the data supported an association with chronic GDNF infusion at
high doses and cerebellar toxicity.
5.5. GDNF: clinical trials and tribulations
5.5.1. Intraventricular infusion of GDNF for PD
In 2003 the results were reported of a randomised double-blind
placebo-controlled trial of intracerebroventricular (ICV) infusion of
GDNF (Nutt et al., 2003). This study recruited 50 patients with mod-
erately advanced L-dopa responsive PD. The study failed to demon-
strate clinical improvement at doses of GDNF sufficient to cause side
effects including weight loss, nausea, anorexia, vomiting, Lhermitte's
phenomenon and hyponatraemia. The authors concluded that the ad-
verse effects associated with treatment confirmed the biological ac-
tivity of GDNF, but the failure to achieve clinical improvement were
likely to be a result of inadequate diffusion of GDNF into the putamen
and substantia nigra. This hypothesis was supported by post mortem
analysis from a trial subject, which showed no evidence of dopami-
nergic neuronal recovery.
5.5.2. Intraputamenal delivery of GDNF
livering GDNF directly to the posterodorsal putamen, which is known to
be the site of greatest dopaminergic neuronal depletion in PD. Gill et al.
undertook the first open-label studyof continuous intraputamenal deliv-
ery of GDNF via stereotactically-placed microcatheters attached to a sub-
cutaneous infusion pump placed in the anterior abdominal wall (Gill et
al., 2003). This study enrolled five patients with symptoms poorly con-
trolled by medical treatment. All five patients demonstrated improve-
ment in both clinical and18F-dopa PET imaging parameters. At twelve
months a significant reduction in Unified Parkinson's Disease Rating
Scale (UPDRS) motor scorewas evident, alongwitheliminationof severe
capacity in the putamen and SN.
All patients entered into a 12 month extension study which
resulted in sustained improvement without serious adverse effects
(Patel et al., 2005). However, T2 MR imaging revealed evidence of in-
creased signal at the catheter tips which may have been attributable
to high infusion flow rates. Consequently the infusion parameters
were maintained at a maximum of 14.4 μg/putamen/day correspond-
ing to a volume of 144 μl. One patient receiving unilateral infusion of
GDNF died of causes unrelated to the study, and post mortem exam-
ination confirmed that infusion of GDNF into the posterodorsal puta-
men resulted in a marked increase in tyrosine hydroxylase-positive
nerve fibres, and possibly neuronal sprouting in the SN (Love et al.,
A second open-label study by Slevin et al. enrolled ten patients,
utilising different catheters, infusion parameters and dosing to the
first study (Slevin et al., 2007). Despite these differences all patients
in this study showed reductions in UPDRS scores as well as improve-
ments in postural stability, dyskinesiae and end-of-dose fluctuations.
Success in these two open-label studies led to the commencement of
a randomised controlled trial.
5.5.3. A multicentre randomised controlled
trial of intraputamenal GDNF infusion for PD
Thirty-four patients were randomised to receive either bilateral
infusions of GDNF into the putamen at a dose of 15 μg/putamen/day
or placebo (Lang et al., 2006). The primary end-point was changes
in the UPDRS motor score in the “off” state, and secondary
end-points included18F-dopa PET, dyskinesia ratings and quality of
life analysis. At six months, patients receiving GDNF had failed to
demonstrate the predetermined level of clinical improvement re-
quired to achieve statistical significance despite improvements in
PET imaging parameters. Furthermore, neutralising antibodies were
detected in 3 patients and a further 3 patients developed serious
device-related adverse effects.
The disappointing results of this randomised study taken in con-
junction with the six-month toxicity study in primates (Hovland et
al., 2007) led to withdrawal of GDNF and cessation of clinical trials.
Technical variations in catheter delivery and design were suspected
to have contributed to the failure of the phase II trial following the suc-
cess of open-label studies. In order to either confirm or refute this hy-
pothesis Salvatore et al. analysed the distribution of125I-GDNF in the
putamen of rhesus monkeys when delivered using the same delivery
system as in the phase II study (Salvatore et al., 2006). Analysis of
GDNF distribution within the putamen by immunohistochemistry re-
vealed significant variability, with the majority of GDNF restricted to
the immediate vicinity of the catheter tip. The authors concluded that
when translated to the human putamen, the bioavailability of GDNF
S.J. Allen et al. / Pharmacology & Therapeutics 138 (2013) 155–175
would have been limited to 2–9% of the putamenal volume. These
findings supported that hypothesis that point source concentration of
GDNF might explain the failure to achieve the expected clinical
5.6. Direct delivery of GDNF to the
brain — current research and future possibilities
5.6.1. CED of GDNF protein
The evidence from the study by Salvatore et al., which implicated
technical issues related to a suboptimal drug delivery protocol in the
failure of the phase II trial (Salvatore et al., 2006) has led to significant
research interest in convection-enhanced delivery (CED). CED de-
scribes the direct delivery of drugs to the brain through ultrafine
intraparenchymal microcatheters. Fig. 6 shows a schematic 2D repre-
sentation of intraparenchymal drug injection compared with CED. By
employing high infusion flow rates and establishing a pressure gradi-
ent at the tip of the catheter, CED confers several potential advantages
over traditional drug injection techniques including homogeneous
distribution throughout large and clinically-relevant volumes of
brain tissue, and reduced tissue trauma.
Image-guided CED of GDNF, using real-time MRI tracking of distri-
bution within the putamen, has been reported in rhesus monkeys
(Gimenez et al., 2011). This technique resulted in widespread distri-
bution of GDNF within the putamen, but was associated with leakage
of GDNF into CSF spaces. For this reason, intermittent delivery of
GDNF was been proposed as a more “physiological” strategy, and
chronic intermittent CED to the putamen has recently been reported
in a large animal model (Bienemann et al., 2011).
5.6.2. CED of GDNF gene therapies
The inevitable clearance of GDNF protein from the brain and re-
quirement for either chronic or intermittent dosing has led to advances
in the development of virally-mediated gene therapies. Significant re-
search effort has been directed at improving the safety and efficacy of
viral vectors for gene therapy in recent years. The evolution of third
generation self-inactivating vectors, tissue-specific promoters, and im-
provements in biodistribution analysis and purification methods have
made clinical translation a realistic ambition (Räty et al., 2008).
Adeno-associated viral vector (AAV2) mediated GDNF gene thera-
py has shown promising results in aged and MPTP-lesioned rhesus
monkeys. Convection-enhanced delivery of AAV2-GDNF into the pu-
tamen and SN of aged primates resulted in increased locomotor activ-
ity, intense GDNF immunoreactivity in the caudate and putamen, and
retrograde and anterograde transport to other brain regions without
significant adverse effects (Johnston et al., 2009).
Importantly a comprehensive safety analysis of AAV2-GDNF deliv-
ery to the substantia nigra or putamen has been undertaken in aged
and MPTP-lesioned primates. This study showed no evidence of im-
mune response, although significant weight loss was observed in
the substantia nigra-treated group (Su et al., 2009).
In a study of intraputamenal CED of AAV2-GDNF in rhesus mon-
keys 3 to 6 months following MPTP lesioning, Kells and colleagues
observed widespread expression of GDNF within the putamen, im-
provement in locomotor function, anterograde transport of GDNF to
the substantia nigra and rescue of dopaminergic neuronal axons
(Kells et al., 2010).
Whilst these preclinical studies confirm the significant potential of
virally-mediated GDNF gene therapy for the treatment of advanced
PD, a recent study using the α-synuclein over-expression model of
PD has raised important issues regarding the interpretation of results
obtained in studies using the 6-OHDA and MPTP models of PD.
Decrassec and colleagues reported that lenti- and adeno-associated
viral vector-mediated delivery of glial cell line-derived neurotrophic
factor into substantia nigra or striatum, was ineffective in preventing
wild-type α-synuclein-induced loss of dopaminergic neurones
(Decrassec et al., 2011). The authors concluded that results from
pre-clinical studies using toxin-induced models of PD must be
interpreted with caution. Certainly, analysis of the effects of GDNF
in mechanistically different models of PD is required before robust
conclusions regarding the translational potential of GDNF gene ther-
apies can be drawn.
Fig. 6. 2D representation of intraparenchymal drug injection compared with CED. Convection-enhanced delivery (CED) differs in both technique and technology to conventional
methods of drug injection. By utilising ultrafine microcatheters, meticulous implantation techniques and establishing a pressure gradient at the tip of the catheter, CED facilitates
homogeneous drug distribution through large and clinically-relevant brain volumes. In contrast, local injection methods are associated with significant reflux of drug, tissue trauma
and inhomogeneous drug distribution which is fundamentally reliant on diffusion rather than bulk-flow through the interstitial spaces of the brain.
S.J. Allen et al. / Pharmacology & Therapeutics 138 (2013) 155–175
5.7. BDNF as a possible therapy for PD
Although GDNF is widely accepted to be one of the best candidates
as a therapeutic approach for PD, the neurotrophin BDNF has also
been investigated as a credible alternative, although only at a preclin-
ical stage. The rationale and some of the associated problems are
presented here; for further detail on this and other neuroprotective
agents which may promote an increase in BDNF see the review by
Fumangali and colleagues (Fumagalli et al., 2006).
BDNF is expressed in the rat substantia nigra and ventral tegmen-
tal area (Gall et al., 1992; Seroogy et al., 1994) and anterogradely
transported from the substantia nigra to the striatum (Altar and
DiStefano, 1998). In PD, reduced BDNF mRNA and protein levels
have been found in striatum, substantia nigra, and to a lesser extent
in cerebellum and frontal cortex (Mogi et al., 1999; Parain et al.,
1999; Howells et al., 2000).
An interesting link has become apparent between these reduced
BDNF levels and the deposits of alpha synuclein seen in neurones in
PD. A study in a glioma cell line found that BDNF was induced by
forms, A30P and A53T, which result in familial PD (Kohno et al., 2004).
This interrelationship of BDNF and alpha synuclein increased in
complexity by the finding that aged mice, heterozygous for the TrkB
droxylase immunoreactive fibres than the wild-type, and had a sub-
stantial accumulation of alpha synuclein neuronal deposits in the
substantia nigra (und Halbach et al., 2005). BDNF reductions found in
PD brain may be important therefore in both familial and sporadic
forms of the disease. Whether or not there is subsequent motor dys-
function will likely depend on the extent of substantia nigra cell loss.
In culture, BDNF promotes neurite outgrowth in dopaminergic
neurones (Studer et al., 1996) and is beneficial to their survival,
protecting them against the neurotoxins 1-methyl-4-phenylpyridinium
(MPP) and 6-hydroxydopamine (6-OHDA) (Hyman et al., 1991; Spina
et al., 1992).
The in vivo effect of BDNF has been reported in an MPTP-induced
primate parkinsonian model (Takeda, 1995). BDNF was applied at the
same time as or before the application of MPTP, by osmotic pump into
the cisterna magna. BDNF treated monkeys showed reduced neuronal
loss and had delayed onset of parkinsonism, suggesting a significant
A further study of the effects of BDNF in a rat PD model used
adeno-associated virus (AAV) vector to enable BDNF expression in the
substantia nigra pars compacta (Klein et al., 1999). Six months later a
partial 6-OHDA lesion was carried out ipsilateral to the injection. Al-
though the BDNF did not preserve tyrosine hydroxylase neurons in
the substantia nigra, it did prevent the amphetamine-induced
turning-behaviour which could be seen in lesioned controls. The BDNF
treated animals were also more active and showed more rotational ac-
tivity on the non-lesioned side than controls. However, in a comparison
study between GDNF and BDNF, BDNF did not appear to provide this
benefit (Sun et al., 2005). Similarly, this was a rat model of PD with
nigrostriatal lesion by intrastriatal injection of 6-OHDA, produced after
injection of vector expressing either GDNF or BDNF, in this case using
herpes simplex virus HSV-1. Here, the application of growth factor
was intrastriatal not into the substantia nigra. The authors reported
icits and in protecting dopaminergic neurones and that expression of
both growth factors gave a similar effect to GDNF alone.
By contrast, more recently, in an organotypic culture model of
Parkinson's disease, BDNF was shown to protect dopaminergic cells
from 6-OHDA-induced cell death even when applied after addition
of toxin. In comparison with GDNF, BDNF further showed transcrip-
tional up-regulation of the dopaminergic phenotype, which led the
authors to conclude that BDNF shows more potential as a therapy
than GDNF in the substantia nigra (Stahl et al., 2011).
There is some hope that amelioration of symptoms of PD may be
made possible by administration of BDNF. However, it would be help-
ful to fully understand the relationship between BDNF and alpha
synuclein, and to determine more clearly the importance of area
and mode of application which would be most likely to provide suc-
cess. Whether it will be more or less useful than GDNF, and whether
in the longer term, possible side effects may be more troublesome
seems to be still somewhat open to debate.
6. Alzheimer's disease (AD), NGF and BDNF
AD accounts for between 60% and 70% of dementias; which trans-
lates into about 500,000 AD sufferers in the UK and 5.4 million in the
US, i.e. 1–2% of the population. Associated costs are currently estimat-
ed at approximately £15 billion per year in the UK, and $183 billion in
the US. Risk factors for AD include advanced age, diabetes and obesi-
ty; given the rising percentage of elderly in the population and the in-
crease in obesity and diabetes, we may expect the prevalence of AD to
increase dramatically over the next decade. It is fundamental there-
fore that we identify the initiating changes in the disease in order to
provide new therapies.
AD sufferers present with a progressive decline in mental func-
tion, usually exhibiting early deficits in memory and other cognitive
processes and often difficulties with language. The neuropathology
shows the presence of numerous ‘neurofibrillary tangles’ (bundles
of paired helical filaments of tau protein within the neurones) and ex-
tracellular deposits of the peptide Aβ (termed ‘amyloid plaques’)
(Hardy, 2006). This peptide is derived enzymatically from the much
larger amyloid precursor protein (APP) and is most usually 40 or 42
amino acids in length. The 42 amino acid version of this has been
shown in numerous studies to be more toxic to neurones. In most
cases the neurofibrillary pathology appears first in the transentorhinal
cortex, progressing to the hippocampus and later to various parts of
the neocortex, with pathology directly correlating with stages of de-
mentia (Braak & Braak, 1991). Fig. 7 shows a broad but concise outline
of the neuropathological processes which occur in the AD brain and
possible therapeutic targets to date.
Longitudinal analysis of an asymptomatic population using MRI
shows changes in the basal forebrain and the entorhinal cortex four
years before onset of symptoms (Hall et al., 2008). Later in the disease
process, thinning of the cortical mantle and enlargement of ventricles
demonstrate gross neuronal loss, with specific regions such as the
hippocampus becoming severely atrophied. On average, sufferers
generally have a diagnosable onset of symptoms of 70 years or
above, and live for eight to ten years subsequent to this. The majority
of cases of AD are termed ‘sporadic’, with likely causes due to a com-
bination of risk factors, both genetic and environmental in origin.
There are rare familial cases of AD (FAD) however, which are early
onset autosomal dominant forms of the disease. The symptoms and
neuropathology are similar to that of the sporadic form but usually
occur with a much earlier onset. There are three genes associated
with FAD, APP (which encodes APP) on chromosome 21 and PSEN1
and PSEN2 (which encode presenilin 1 and 2 — either of which
forms the catalytic part of gamma secretase. These latter genes are
present on chromosomes 14 and 1 respectively. Down's syndrome
(Trisomy 21) is caused by an extra chromosome 21 or part of it. In
most cases this results in an amyloid-associated dementia by the
fourth decade. It is assumed that it is the presence of an extra copy
of APP which causes this, because of an increased Aβ formation. Par-
ticular mutations in the three genes can cause FAD, by the production
of increased total Aβ or an increase in the Aβ42:40 ratio (Borchelt et
al., 1996). These mutations have been used to produce transgenic an-
imals which resemble certain aspects of AD (Howlett, 2011). The am-
yloid hypothesis (Hardy & Allsop, 1991) which (in brief) states that
all pathology and symptoms of AD stem from the effects of Aβ, im-
plies that the presence of Aβ is causative of neurofibrillary tangle
S.J. Allen et al. / Pharmacology & Therapeutics 138 (2013) 155–175
production. To date there has been no substantial evidence yet sub-
mitted to challenge this hypothesis convincingly (Goate & Hardy,
6.1. The cholinergic changes in AD
In the late 1970s the loss of cholinergic function in the AD brain
was observed independently by three groups (Bowen et al., 1976;
Davies & Maloney, 1976; Perry et al., 1977). A study of biopsy samples
in the early 1980s showed that cholinergic function, as measured by
choline uptake, acetylcholine synthesis and choline acetyltransferase
(ChAT) activity in early-stage AD patients, was reduced to approxi-
mately a third of normal (Sims et al., 1983). Over 90% of cholinergic
neurones of the basal forebrain are immunopositive for p75NTR in
rat and human (Dawbarn et al., 1988; Allen et al., 1989). In AD
brain the ChBF neurones (as assessed by p75NTR staining) were rela-
tively spared although many were atrophied, with some cell loss
mainly in the posterior region. This was despite a 50–90% loss of cor-
tical ChAT activity. Neuronal preservation was seen particularly in
older patients (Allen et al., 1990), whilst younger patients were
reported as having marked cell loss (Mufson et al., 1989; Allen et
al., 1990). The relative survival of these neurones, coupled with the
beneficial effects seen with NGF administration (see Section 6.5)
supported at this time, a rationale for NGF as a possible therapeutic
approach for support of cholinergic innervation of the cortex and
6.2. The role of neurotrophins in the pathogenesis of AD
The links between the cholinergic changes in AD and changes in
the neurotrophins NGF and BDNF have been underlined by a number
of studies. Following on from Appel's ‘unifying hypothesis’ the con-
cept of a reduction in NGF, produced by the hippocampus and cortex
was explored. However, no reduction in NGF in the cortex or hippo-
campus could be seen by ELISA (Allen et al., 1991), surprisingly
there appeared to be an increase (Crutcher et al., 1993). Despite
this, levels of NGF in the ChBF were decreased (Mufson et al., 1999).
This may be related to the fact that retrograde transport of NGF is
likely to be compromised in AD.This hasbeen shownexperimentally:
125I labelled NGF injected into rodent hippocampus, travels retro-
gradely via the fornix to the ChBF in about six hours, whereas in a
subset of aged rats (Cooper et al., 1994) and in a mouse model of
Down's syndrome (Trisomy 16) (Cooper et al., 2001), a severe loss
of retrograde transport of NGF was apparent.
Later proNGF was shown as not only the dominant form in the
human cortex but the only form which could be visualised
(Fahnestock et al., 2001); the increase seen earlier in AD brain tissue
was therefore in the proform, not the mature form of NGF.
In 2000 a transgenic mouse model of cholinergic loss was generat-
ed (Capsoni et al., 2000). This mouse (AD11) expressed an antibody
to NGF; these antibodies accumulated throughout the adult life,
thus circumventing the developmental problems inherent with NGF
knockouts. These mice had, as expected, a reduction in ChBF
neurones, and there were also measurable deficits in ability to
Fig. 7. A simplified schematic of some of the pathological processes in AD and potential therapeutic targets. This intends to put the neurotrophins in perspective as potential ther-
apeutic targets and does not intend to be comprehensive in terms of pathology or therapeutics. Pathology: Amyloid precursor protein (APP) is cleaved mostly (about 95% of the
time) by alpha secretase (ADAMs (a disintegrin and metalloprotease) enzyme family of secretases either ADAM 9, 10 or 17). This initiates the non-amyloidogenic pathway in
which a C-terminal 83 amino acid peptide is produced. If beta secretase (predominantly BACE1 (beta-site APP-cleaving enzyme)) cuts APP then a C-terminal of 99 amino acids
is produced. Gamma secretase (comprises four proteins including presenilin (mainly PS1)) cleaves C83 to form p3 (3 kDa protein). It cleaves C99 to form a 4 kDa protein (Aβ)
plus APP intracellular domain (AICD). Aβ is usually 40 amino acids long (Aβ40), this is soluble enough to reach the blood vessels where it may be deposited. Infrequently Aβ42
is formed, which fibrillises quickly and therefore deposits as amyloid plaques before it can be cleared. This form is thought to be the more toxic, especially inside neurones, as it
causes production of reactive oxygen species which destabilises membranes and forms toxic products. Amyloid plaques stimulate inflammatory cytokine production which causes
further neuronal damage. Tau, which normally binds microtubules, becomes hyperphosphorylated by kinases (e.g. glycogen synthase kinase 3; GSK3β); this is thought to cause
them to fall away from the microtubules and aggregate into tangles. Therapeutic targets: T1: CEIs; T2: BACE1 inhibitors; T3: Vaccines to clear soluble and aggregated Aβ; T4:
Gamma secretase inhibitors; T5: De-aggregators of tau or kinase inhibitors (e.g. GSK3β inhibitors); T6: NGF or BDNF protein or small molecule agonists/modulators; T7:
non-steroidal anti inflammatory drugs (NSAIDs) to decrease inflammation; T8: ApoE enhancers to clear Aβ; T9: agents to reduce mitochondrial stress; T10 stimulation of hippo-
campal pathways by deep brain stimulation; and T11 antibiotics to prevent infection which leads to glial activation and TNFα release.
S.J. Allen et al. / Pharmacology & Therapeutics 138 (2013) 155–175
perform memory tasks. However, the antibody produced by the AD11
mice has been described as binding to mature NGF with greater affin-
ity than to proNGF, thus producing an increase in the proNGF/NGF
ratio. It is presumably because of this that the mice unexpectedly
also presented with an AD-like pathology with amyloid plaques, cor-
tical cell loss, but also deposition of hyperphosphorylated tau in cor-
tical and hippocampal neurones. In a subsequent study effects of
intranasal administration of NGF (1.2–12 μg on alternating days) re-
stored the number of ChAT-immunopositive neurones to normal, re-
versed deposition of hyperphosphorylated tau and strikingly reduced
amyloid plaque numbers. Administration of the cholinesterase inhib-
itor (CEI) galantamine (3.5 mg/kg daily) largely removed amyloid
plaques, yet had no effect on deposition of hyperphosphorylated tau
(Capsoni et al., 2002).
More recently the mechanisms involving TrkA and p75NTR were
further investigated. The AD11 mice were crossed with a p75NTR de-
ficient mouse to form the AD12 mouse; this rescued the cholinergic
function and prevented amyloid deposition but unexpectedly caused
an increase in tau-related pathology (Capsoni et al., 2010). Evidently
there is a definite relationship between the AD pathology and both
the TrkA and p75NTR signalling. Interestingly, a transgenic mouse
(TgMNAC13), which expresses a neutralising anti-TrkA antibody,
was shown to produce a deficit in cholinergic function and amyloid-
but not tau-associated pathology (Capsoni et al., 2010).
There is also justification for suggesting a role for BDNF in the
pathogenesis of AD, since BDNF mRNA and protein are substantially
reduced in the AD cortex and hippocampus (Phillips et al., 1991;
Connor et al., 1997; Ferrer et al., 1999; Hock et al., 2000; Holsinger
et al., 2000; Peng et al., 2005). TrkB is also reduced in frontal and tem-
poral cortex, but only the catalytic form (145 kDa) not the truncated
forms (95 kDa) (Allen et al., 1999; Ferrer et al., 1999). Given its im-
portance in LTP and memory, BDNF-associated therapy also seems
appropriate (see Section 6.6).
6.3. Current therapies for AD
CEIs have been used to treat AD patients with partial success, since
a study in 1986 first showed the benefits of tetrahydroamino acridine
(THA, Tacrine) (Summers et al., 1986). Second generation CEIs,
donepezil (Aricept™), galantamine (Reminyl™) and rivastigmine
(Exelon™) are now recommended in the UK (TA217 March 2011 Na-
tional Institute for Health and Clinical Excellence (NICE) technology
appraisal) as options for managing mild and moderate AD. These
work at the cholinergic synapse, inhibiting acetylcholinesterase
(cholinesterase)-mediated hydrolysis of acetylcholine and potentiat-
ing its signal.
Memantine, an uncompetitive antagonist at the NMDA receptor, is
recommended as an option for management of severe AD and for
managing moderate AD for sufferers unable to take CEIs. Memantine
inhibits the prolonged influx of Ca2+, and thus prevents excitotoxicity
due to excess glutamate. Both types of therapy contribute to mainte-
nance of ChBF–hippocampal interaction, which apparently helps to
prolong cognitive function in perhaps a half of patients given the
medication. However, the onslaught of the pathological process is
such that within one to two years the benefits usually cease.
These current therapies, although extremely useful, cannot hope
to contend with the alarming increase in the numbers of AD sufferers
world-wide. To this end there have been a variety of other ap-
6.4. New therapeutic targets in AD
The use of growth factors such as NGF and BDNF has been pro-
posed and preclinical and clinical studies are providing a degree of
encouragement. However, the disease itself has multiple facets and
therefore offers a number of possible therapeutic targets. Fig. 7
attempts to put growth factor therapy in the context of some of the
other potential solutions. The dominant approach, understandably,
has been to try to clear Aβ from the brain or to prevent its production.
Clinical trials for (anti-Aβ) vaccines, beta-site APP-cleaving enzyme 1
(BACE1) antagonists or gamma secretase inhibitors, despite having
had setbacks are still likely to be popular choices. The inhibition of
gamma secretase is proving problematic as it has many substrates,
one of which is notch, and unfortunately notch signalling defects
are themselves implicated in various diseases. For example, a
gamma secretase inhibitor Semagacestat (Eli Lilly) Phase III trial
was stopped in 2010 as results showed a more rapid decline in cogni-
tive function of patients and an association with a greater risk of skin
cancer. It may be possible to overcome this problem with increased
selectivity; BMS-708163 (Bristol Myers Squibb) has fewer side effects
than Semagacestat, probably due to its increased selectivity for APP
over notch. In a Phase I trial it showed substantial reductions (up to
60%) in CSF Aβ40/42 after a month.
Currently there are approximately 40 active FDA registered clini-
cal trials of drugs for treatment of AD, some of which are novel, and
it may well be that a number of different approaches are needed. Un-
fortunately, whilst the FAD mutations do not fully represent the AD
pathogenic mechanisms underlying the sporadic forms of the disease,
they seem at present to provide foundation for our best mouse
models. There are other genetic leads however, the presence of the
APOE4 allele alone represents perhaps a 20–25% additional risk factor
for the disease. This polymorphism may have a number of disadvan-
tages, decreased clearance of Aβ and the reduced ability for recovery
after neuronal damage being perhaps two of the most problematic.
Recent studies using retinoid X receptor (RXR) (Cramer et al., 2012)
and liver X receptor (LXR) agonists (Terwel et al., 2011) to increase
apolipoprotein E (apoE) expression seem to give hope for Aβ clear-
ance, but the question remains whether increasing apoE in those pa-
tients with the E4 polymorphism will help or hinder any recovery.
In an interesting parallel with PD therapies, a new approach to
stimulation of the hippocampal pathways is being tested in AD pa-
tients. In a Phase I trial from 2005 to 2008, Andres Lozano's team in
Toronto used bilateral chronic deep brain stimulation (DBS) of the
fornix (the connection between the ChBF septal nucleus and the hip-
pocampus) for 12 months in six patients with mild AD. PET scans
showed reversal of the impaired glucose utilisation in the cortex,
and cognitive examination suggested some slowing in the rate of cog-
nitive decline. This is being followed up with a second Phase I feasibil-
ity study which will be double blind, randomised controlled, to
evaluate safety, efficacy and tolerability. This will be in subjects diag-
nosed with probable mild AD.
6.5. NGF protein as a therapeutic option for AD
From the 1980s onwards, a number of in vitro studies revealed the
importance of NGF for cholinergic function, its ability to enhance the
One established model of cholinergic loss is the fimbria–fornix lesion in
which the connection between the basal forebrain cholinergic septal
nucleus and the hippocampus is severed, which prevents retrograde
transport of growth factors to the ChBF. In rodents and primates this
leads to ChBF atrophy, shrunken cholinergic neurones, loss of choliner-
gic markers and results in memory deficits. During the 1980s and 90s a
number of studies showed that ICV administration of NGF prevented or
even reversed these changes (Will & Hefti, 1985; Williams et al., 1986;
Kromer, 1987). Interestingly, a subgroup of aged rats with cholinergic
deficits and which performed badly in classic memory tests such as
the Morris water maze, were found to perform as did the younger rats
after receiving ICV administration of NGF (Fischer et al., 1987).
In the 1990s a report was published following the first ICV infu-
sion of NGF into an early onset AD patient (Olson et al., 1992) in
S.J. Allen et al. / Pharmacology & Therapeutics 138 (2013) 155–175
which 6.6 mg purified mouse NGF was given over a period of three
months. An increase was seen in [11C]-nicotine binding in frontal
and temporal cortex and a persistent increase in cortical blood flow.
Verbal episodic memory was improved after a month, although
other cognitive tests were not. Subsequently, the final result of infu-
sion in three patients was reported in 1998 by the same group
(Eriksdotter et al., 1998). Three months after treatment, an increase
in [11C]-nicotine binding, indicating an increase in cholinergic synap-
ses, was seen in the two patients given higher doses, and slow-wave
cortical activity measured by electroencephalogram was reduced.
Some neuropsychology tests showed slight improvement, however
side effects were observed, including weight loss and also an NGF
dose-related back pain. Rodent and primate studies subsequently re-
vealed the presence of a reversible (dose-related) non-malignant
Schwann cell hyperplasia attached to the medulla and spinal cord
(Day-Lollini et al., 1997; Winkler et al., 1997). It is likely that this
was caused by the high levels of NGF infused into the ventricles,
with consequent overspill into the surrounding tissues. Since the ef-
fect was via p75NTR, as these hyperplastic cells do not express TrkA,
it is possible that either judicious administration of NGF into the pa-
renchyma, or the use of a TrkA agonist or modulator may provide
benefit without this particular drawback.
Other routes of NGF protein administration have been explored.
Fibroblasts transfected to produce mature NGF, implanted into the
basal forebrain, have been shown to rescue cholinergic function in
rats after fimbria–fornix lesion (Blesch et al., 2001) and in aged
cholinergic-deficient rhesus monkeys (Tuszynski et al., 1998;
Smith et al., 1999). However, it was noted that in these studies it
took several months for cortical cholinergic terminal density to in-
crease. Retrograde transport of radiolabelled NGF to the cholinergic
basal forebrain has previously been reported following both nasal
administration (Thorne & Frey, 2001) and injection into the rat ol-
factory bulb (Altar & Bakhit, 1991). More recently there have been
two studies using intranasal application of NGF. In one, NGF was
delivered intranasally to rats after traumatic brain injury (TBI).
This attenuated the TBI-induced Aβ deposition in the cortex and
hippocampus and improved functional outcome (Tian et al.,
2012). In a second study a mutant form of NGF (P61S/R100E) was
delivered intranasally to a transgenic mouse model (tgAPPxPS1).
This arrested the progress of neurodegeneration and behavioural
deficits of the AD mouse (Capsoni et al., 2012). The additional
novel aspect of this study was that since NGF protein may trigger
hypersensitity to pain by binding to the TrkA and p75NTR recep-
tors, use of an NGF mutant which does not trigger pain pathways,
in particular the phospholipase Cγ (PLCγ) pathway, may circum-
vent this particular problem. This relates to previous work carried
out by this group on R100W, an NGF mutation which causes pain
hyposensitivity in a Swedish cohort (HSAN V; Hereditary Sensory
Autonomic Neuropathy Type V).
The concept of NGF protein administration as an AD therapeutic
was translated to a Phase I clinical trial initiated in 2001 (Tuszynski
et al., 2005). Mark Tuszynski's team at the University of California
surgically implanted eight early stage AD patients with their own fi-
broblasts which had been modified (using a Moloney leukaemia
viral vector) to secrete mature human NGF. These implants of modi-
fied cells were injected at five sites along the ChBF (each projection
of the basal nucleus is about 2 cm long, front to back). Unfortunately,
two of the patients suffered operation-associated haemorrhages;
however, the six remaining patients, monitored for up to 22 months
post operation, showed no treatment-related ill effects. Mini-mental
status examination scores (MMSE), which were declining at six
points per year on average before the operation, slowed to half this
rate of decline post operation. Positron emission tomography (PET)
analysis of the four subjects given most cells at implantation, showed
a marked increase in cortical fluorodeoxyglucose (FDG-PET) uptake in
cortical areas innervated by the ChBF, six to eight months after
implantation. Up to 18 months after treatment, five of the six subjects
showed MMSE scores which were either improved (n=2), unchanged
(n=1) or showed a minor decline (n=2). Because the patients were
each only at an early stage in the disease a definitive diagnosis had
not been possible; in fact one of the eight original patients that died
after five weeks was seen to have Lewy body disease, combined with
the ChBF, which would explain the increased cortical FDG-PET results
The results from this clinical trial provided impetus for a Phase I
trial (Ceregene Inc., San Diego) testing the safety, tolerability and pre-
liminary efficacy of adeno-associated virus (AAV)-mediated delivery
of NGF (CERE-110; AAV-NGF) by stereotactic injection into the
brain of six patients. The results of this were reported in 2008
(Bishop et al., 2008). Subjects had MMSE scores between 16 and 26,
with probable mild-to-moderate AD. They received CERE-110 injec-
tions, at two sites alongside the ChBF, of either 8×109or 4×1010vec-
tor genomes. Safety, cognition and FDG-PET (fluorodeoxyglucose
(18F) PET) were assessed for one year, during which time no
CERE-110 related adverse events occurred. Six months post-therapy,
significant increases in glucose uptake were seen in several cortical
regions by PET scan. Cognitive tests showed on average small de-
creases over the year post surgery (e.g. a decline on the MMSE of
1.7–2.1 points). The conclusion was that this method was safe to pro-
ceed to Phase II trials. Ceregene, in collaboration with the Alzheimer's
Disease Cooperative Study (ADCS), is now recruiting for a Phase II
multi-centre clinical trial in approximately 50 patients with mild to
moderate AD, to be conducted at 10 sites in the US. This will be
randomised, with half the patients undergoing sham/placebo surgery,
and all patients monitored for at least two years after surgery.
6.6. BDNF protein as a therapeutic option for AD
There have been a number of preclinical studies which suggest
that replacement of BDNF may ameliorate both age-related and
In 1995 Mamounas and colleagues showed that BDNF given
intraparenchymally, prevented degeneration of serotonergic axons by
the serotonergic neurotoxin p-chloroamphetamine (PCA) (Mamounas
et al., 1995). BDNF was infused continuously by osmotic minipump
into the frontoparietal cortex of rats with a PCA subcutaneous injection
after 7 days, followed by 14 days of further BDNF infusion. This sub-
stantially decreased the loss of serotonin axons in a 3 mm diameter re-
gion surrounding the cannula tip, highlighting the problem of BDNF's
low rate of diffusion in the brain. Delivery of BDNF into the brain (as
mentioned in Sections 4.1 and 4.2), to overcome the problems of mini-
mal brain penetration with intraventricular or intrathecal administra-
tion, provides a substantial challenge.
Other methods of delivery have therefore been investigated. Cog-
nitive deficits in a rat model of synaptic lesion after administration of
botulinum neurotoxin type B into the entorhinal cortex, were partially
rescued by adenoviral-mediated BDNF gene overexpression (Ando et
tricle (thus allowing infusion to both sides of the brain) showed sub-
stantial protection from Aβ when given concurrently (Arancibia et al.,
In a series of impressive in vivo experiments, BDNF was given to
rodents and primate models of advanced age and AD by different
routes. In each case, disease-related symptoms were improved
(Nagahara et al., 2009). The target was the entorhinal cortex, partly
because of its early and marked impairment in AD and partly as it is
a major source of input to the hippocampus and is important in mem-
ory function; BDNF and TrkB are both highly expressed in this region.
In AD transgenic mice, BDNF gene delivery administered after dis-
ease onset, despite no obvious increase in clearance of Aβ or amyloid
S.J. Allen et al. / Pharmacology & Therapeutics 138 (2013) 155–175
plaques, reversed synapse loss and restored cell signalling, learning
and memory. At six months lentiviral vectors expressing BDNF
under different promoters were injected into the entorhinal cortices
of J20 transgenic mice. These mice have APP mutations at both the
β- and γ-secretase sites. They produce cortical amyloid plaques by
2–3 months with cell loss in the entorhinal cortex, and show cogni-
tive decline by 6–7 months of age. After one month, spatial memory
was tested and found to be significantly improved. Transport of BDNF
into hippocampal regions CA1–3 and dentate gyrus could be seen,
with recovery of synaptic markers in the entorhinal cortex and dentate
gyrus. Whereas overexpression of mutant APP causes changes in path-
ways related to oxidativestressand lipid metabolism, BDNF gene deliv-
In a rat model of ageing (24 months), a reduction occurs in
entorhinal-associated cognitive function, which is not cell-death re-
lated. In aged Fischer rats, spatial learning and memory were assessed
before and after a 28 day infusion of BDNF into the medial entorhinal
cortex (120 ng per day per side). BDNF infusions significantly im-
proved spatial learning and memory in water maze performance
(Nagahara et al., 2009).
In rhesus monkeys, radiofrequency lesions of the perforant path
were performed bilaterally, and lentiviral vector expressing BDNF
was injected into the right entorhinal cortex, whereas vector without
BDNF was injected into the left side. Stereological quantification after
perforant path lesions resulted in the preservation of 85% of neurones
in the presence of BDNF compared with 54% without BDNF (Nagahara
et al., 2009).
In a primate model of age-related neurodegeneration there is cog-
nitive decline and neuronal function deficits without extensive cell
death. Aged monkeys showed significantly impaired visuospatial
learning compared to young monkeys as characterised by a discrimi-
nation task, which is sensitive to temporal lobe function. Bilateral in-
jections of lentiviral vector expressing either BDNF or GFP were given
to aged monkeys in four locations in the medial temporal cortex,
targeting neurones projecting to the hippocampus. Monkeys given
BDNF showed a significant improvement in performance compared
with those which were not. Elevated levels of BDNF were found in
the hippocampus, suggesting anterograde transport (Nagahara et al.,
BDNF administration in each of these models prevented cell death
and reduced memory deficits. This establishes an excellent rationale
for BDNF delivery to the entorhinal cortex as a means for treating ento-
rhinal and hippocampal degeneration in AD. However, apart from the
distribution problems associated with infusion of BDNF, the BDNF sig-
nalling pathways are central to many functions within the brain, and
its production and release are highly controlled. It is debated as to
whether detrimental effects may occur due to hyperexcitability if ex-
cess amounts of BDNF are infused into the brain (Murray & Holmes,
2011). A number of studies have shown a link between increased
BDNF protein levels and seizure, for instance, after seizure BDNF in-
creases; whereas seizures are lessened in response to kindling in
BDNF null mice (Kokaia et al., 1995).
Alternatively, various agents have been shown to increase BDNF ex-
pression in the brain. Memantine, a medium-affinity uncompetitive
N-methyl-D-aspartate receptor antagonist, caused a dose-related in-
crease in BDNF mRNA and protein levels in rat brain, and also TrkB re-
ceptor levels in the cortex (Marvanova et al., 2001). In simian
immunodeficiency virus-infected rhesus macaques, which are a model
of HIV neurodegeneration with evident brain dysfunction and patholo-
gy at 3–5 monthsafterinfection, memantineadministration resultedin
upregulation of BDNF mRNA and protein expression (Meisner et al.,
2008). In addition, oral administration of the CEI donezepil to AD pa-
tients was shown to result in an increase of BDNF levels in serum
(Leyhe et al., 2008). These results need to be understood in the context
of the known effects of these drugs in AD patients.
Below is a summary of some recent studies in which neurotrophin
receptors have been targeted and the effects of small molecules were
6.7. NGF and BDNF receptors as therapeutic targets
The neurotrophins activate their respective Trk receptors in the
brain, usually with beneficial effects. In addition they can also bind
p75NTR with contextually-related effects. ProNGF and proBDNF
have been shown to bind sortilin and p75NTR to initiate apoptosis.
Each of these receptors present putative targets for therapy in AD.
6.7.1. The p75NTR receptor as therapeutic target
Depending on context, p75NTR can be associated with cell death;
it is expressed by the spinal motor neurones which degenerate in ALS,
and also on the ChBF neurones and some hippocampal cells which de-
generate in AD. Frank Longo's group at Stanford are investigating the
small molecules LM11A-24, a caffeine derivative, and LM11A-31, an
isoleucinederivative,which are antagonists at p75NTR withnanomolar
of the neurotrophin (loop 1)-p75NTR interacting domain (Massa et al.,
2006). LM11A-24 has been shown to promote hippocampal neurone
survivaland to protectoligodendrocytes fromproNGF-induced apopto-
sis via p75NTR. LM11A-24 prevented p75NTR-dependent motor neu-
rone death induced by NGF or by spinal cord extracts from
SOD-1G93A mutant mice (Pehar et al., 2006).
Further to this they havebeenshown toinhibitAβ-initiated neuritic
dystrophy and pyramidal cell death in hippocampal slice cultures, and
inhibit kinases, such as cdk5 and GSK3β, which are implicated in AD
pathogenesis. This resulted in reduced tau phosphorylation, and pre-
vention of AKT and CREB inactivation. LM11A-31 also blocked
Aβ-induced deficits in hippocampal LTP (Yang et al., 2008).
6.7.2. TrkA as therapeutic target
NGF mimetics, described in 2000, activated ERK and AKT and pro-
duced neurotrophic effects in vitro. These were structures composed
of dimers of a TrkA binding loop of NGF (Xie et al., 2000). Other sim-
ilar mimetics have since been developed with in vitro neurotrophic
activity (Zaccaro et al., 2005; Peleshok & Saragovi, 2006).
Additionally, a small-molecule agonist at TrkA was identified by
a high-throughput screening assay. Gambogic amide (present in
resin exuded from the Garcinia hanburryi tree), interacts at the
domain closest to the membrane of TrkA and induces receptor
dimerisation, phosphorylation and signalling. It also prevents
glutamate-induced neuronal death and initiates neurite outgrowth
in PC12 cells. Gambogic acid administration decreases size of in-
farcts produced in a transient cerebral artery occlusion model of
stroke and also reduces neuronal death seen in mice lesioned with
kainic acid (Jang et al., 2007). It has been previously identified as
an anti-cancer agent and triggers apoptosis after binding to the
transferrin receptor. Its therapeutic use in such diseases as AD may
therefore depend on its ability to bind TrkA more tightly than the
transferrin receptor in vivo.
Most recently the characterization of small molecule NGF agonists
with nanomolar affinity at TrkA was described (Scarpi et al., 2012). Com-
TrkA crystal. Some compounds were active in NGF-dependent assays;
one of these, MT2, displaced NGF binding to TrkA, induced TrkA auto-
phosphorylation and downstream signalling and promoted differentia-
tion of serum-starved PC12 cells. It is suggested by the researchers that
the NGF binds to a TrkA monomer and does not dimerise the receptors;
MT2 may therefore induce conformational change which results in stim-
ulation of receptor autophosphorylation. Interestingly, strong TrkA phos-
phorylation was seen at residue Tyr490, but not the other tyrosine
residues i.e. 674/5 and 785. This leads to differential selection of down-
stream pathways and may explain why trophic activity such as survival
S.J. Allen et al. / Pharmacology & Therapeutics 138 (2013) 155–175
of serum-starved PC12 cells and rescue of organ cultured rat hippocam-
pus with NGF-deprivation was found to be strong, whereas differentia-
tion responses such as neurite outgrowth were weak. This line of
research looks promising and hopefully will lead to drug candidates
(Scarpi et al., 2012).
6.7.3. TrkB as therapeutic target
Agonists at the TrkB receptor have also been described by Frank
Longo's group for use in AD and Rett syndrome. Using a BDNF
loop-domain pharmacophore, in silico screening and in vitro screen-
ing, allowed identification of small molecules with nanomolar activity
at TrkB. The small molecule LM22A-4 was selected and was shown to
prevent neuronal degeneration with similar efficacy to BDNF. After
administration in vivo to rats with traumatic brain injury, improve-
ment was seen in motor learning (Massa et al., 2010).
LM22A-4 was also evaluated in the Rett syndrome mouse model,
the Mecp2 mutant. This mouse has a breathing dysfunction; four
weeks treatment produced an increase in TrkB phosphorylation in
the medulla and pons and restored normal breathing frequency
(Massa et al., 2010). The researchers suggest, as do Scarpi and col-
leagues in their discovery of NGF mimetics (Scarpi et al., 2012), that
the effects are due to conformational changes in the receptor which
may not exactly reproduce the effects of the ligand. They suggest
that further studies are required making comparison between signal-
ling pathways between these compounds and BDNF and NT4.
Additionally, certain flavonoids, found in abundance in many fruits
and vegetables, have been suggested as neuronal protectants and
memory enhancing agents (Spencer, 2008). 7,8-dihydroxyflavone
was identified as a TrkB agonist, able to activate downstream signal-
ling, and protect neurones in a TrkB-dependent manner, from apopto-
sis. It was shown to be protective in mice models of toxicity by kainic
acid, stroke and PD (Jang et al., 2010). Furthermore, by systemic ad-
ministration in mice (at 5 mg/kg i.p.) it enhanced acquisition of fear
and its extinction (Andero et al., 2011) and prevented immobilisation
stress-impaired acquisition of long term memory. Its use may there-
fore be applicable therapeutically for post-traumatic stress disorder
(Andero et al., 2012).
Most recently the effect of 7,8-dihydroxyflavone was observed in
the 5XFAD transgenic mouse model of AD [with APP mutations
KM670NL, I716V, V717I and PS1 mutations M146L/L286V]. These
mice have reduced BDNF and TrkB levels and phosphorylation of
TrkB receptors. 7,8-Dihydroxyflavone restored the deficient TrkB sig-
nalling, although not BDNF levels, and rescued memory deficit in the
spontaneous alternation Y-maze task. 5XFAD mice also have in-
creased BACE1 levels and activity, and increased production of Aβ40
and Aβ42. These were all prevented by administration of the flavo-
noid (Devi & Ohno, 2012).
In addition, 7,8-dihydroxyflavone when given to the Rett syn-
drome mouse model, the Mecp2 mutant, increased life span, delayed
body weight loss, improved breathing and increased ability to take
voluntary exercise (Johnson et al., 2012).
7. Summary and conclusion
Neurodegenerative diseases represent a major socioeconomic
burden and unimaginable misery for millions of sufferers and their
families around the world. With an ageing population, the number af-
fected is set to rise even further, creating an urgent need for thera-
peutic strategies which can reverse or halt the degenerative process.
GDNF, BDNF and NGF are important trophic factors in adulthood as
well as development, and each is required by certain subsets of
neurones for optimum function. A reduction in level of one or more
of these proteins may be responsible for at least some of the symp-
toms of AD, PD, ALS, HD and Rett syndrome. Each of the factors has
been used as a neurorestorative therapy in some form or other in clin-
ical trials: GDNF in PD by ICV or putaminal infusion, BDNF given
intrathecally in ALS, NGF given in AD as an infused protein, and
implanted cells secreting NGF, or by AAV delivery. The delivery of
proteins into the human brain has inherent difficulties, and it is likely
that the low success rate has been largely due to the protein not
reaching the target at a sufficient concentration.
The results from AAV administration should soon provide an an-
swer as to whether improvements are similar to those seen in animal
models and whether they can be sustained over a long period. It is ob-
vious that treatment will need to be given as early as possible, but
these trials may give some indication as to whether the growth fac-
tors can halt or even reverse the disease process.
Ultimately however, this form of treatment will not be able to sat-
isfy the ever increasing global demand for therapy for these diseases.
New modifying agents such as the p75NTR antagonists and Trk ago-
nists or modifiers may provide new hope for cheap and accessible
treatment. However, many existing drugs and dietary supplements
are now being tested for their neuroprotective effects, and since
these have already been available for public consumption this may
provide more rapid access to a therapeutic hope for the rising number
of people affected by these most distressing diseases.
Conflict of interest statement
The authors declare that there are no conflicts of interest.
This work was supported in part by the Royal College of Surgeons
of England, Bristol Research into Alzheimer's and Care of the Elderly,
UK (BRACE), the Dunhill Medical Trust, Alzheimer's Society, UK, and
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