brain derived neurotrophic factor and neurodegeneration

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Brain derived neurotrophic factor and neurodegeneration Cristian L Achim & Michael G White University of Pittsburgh School of Medicine, BST-S406, Dept. of Pathology, 200 Lothrop St., Pittsburgh, PA 15213, USA Brain derived neurotrophic factor (BDNF) was the subject of over one hundred patents in the past three years. The majority of these patents propose new methods to increase its bioavailability and clinical benefit (that is yet to be defined in the adult human nervous system). The major limitation in the current neurotrophic factor (NTF) research, impairing relevant comparisons of preclinical results, is the incomplete methodo- logical standardisation in measuring the effects of BDNF in experimental models. Nevertheless, BDNF has clearly emerged as the leading candidate to become the pluripotent neurotrophic factor that may soon be either the prime factor or at least a universal adjuvant in future neuroregenerative therapies. Furthermore, our current understanding of BDNF receptor biology, especially of the high affinity tyrosine kinase B (trkB) receptor, has spurred research targeting the design of more potent BDNF-like synthetic ligands. Finally, new strategies are currently explored to increase the target availability of BDNF and expand its biological life. Keywords: brain derived neurotrophic factor, neurodegeneration, neurotrophins, regeneration, therapy Exp. Opin. Ther. Patents (1999) 9(12):1655-1664 1. Introduction BDNF research is second only to its neurotrophin relative, nerve growth factor (NGF). It is now accepted that the pluripotent BDNF and its high affinity receptor trkB are widely distributed both in the developing and mature nervous system. For this review, we read the description of BDNF (but not exclusively) oriented patents submitted between 1996 and the first half of 1999 and attempted to put them in the context of the abundant litera- ture on the subject (over 250 Medline references in the first six months of 1999!). We believe that neurotrophin research is, arguably, the hottest area in neuroscience today and BDNF will probably be the subject of a signifi- cant number of discoveries about mechanisms of neurodegeneration and potential therapeutic interventions. Our review will focus on the major directions of research and principles of therapeutic intervention rather than an exhaustive account of progress in the field. For this latter purpose, the reader will be directed to some excellent reviews. The majority of bibliography is relevant to the principles discussed in the patents reviewed and may not always be the ultimate report on the topic in general. Although we tried to just summarise the patents or scientific reports outlined in this review and then present our opinion at the end, we sometimes felt that it would be pertinent to comment 1655 1999 © Ashley Publications Ltd. ISSN 1354-3776 Review 1. Introduction 1.1 The almost good news 1.2 The good news 2. BDNF: the basics 2.1 BDNF in human brain degeneration 2.2 New functions attributed to BDNF 2.3 Clinical targets 3. Recent technical developments 3.1 New experimental models 3.2 New methods to analyse BDNF and trkB activities 4. Designing BDNF based therapies 4.1 In vivo delivery 4.2 Bioengineering and bioavailability 4.3 Natural resources 5. Expert opinion 5.1 BDNF activities 5.2 From the research laboratory to the clinic 5.3 New therapeutic designs Acknowledgements Bibliography Patents http://www.ashley-pub.com Expert Opinion on Therapeutic Patents

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Achim & WhiteBrain derived neurotrophic factor and neurodegeneration Brain derived neurotrophic factor and

neurodegeneration

Cristian L Achim & Michael G White

University of Pittsburgh School of Medicine, BST-S406, Dept. of Pathology,200 Lothrop St., Pittsburgh, PA 15213, USA

Brain derived neurotrophic factor (BDNF) was the subject of over onehundred patents in the past three years. The majority of these patentspropose new methods to increase its bioavailability and clinical benefit(that is yet to be defined in the adult human nervous system). The majorlimitation in the current neurotrophic factor (NTF) research, impairingrelevant comparisons of preclinical results, is the incomplete methodo-logical standardisation in measuring the effects of BDNF in experimentalmodels. Nevertheless, BDNF has clearly emerged as the leading candidateto become the pluripotent neurotrophic factor that may soon be either theprime factor or at least a universal adjuvant in future neuroregenerativetherapies. Furthermore, our current understanding of BDNF receptorbiology, especially of the high affinity tyrosine kinase B (trkB) receptor, hasspurred research targeting the design of more potent BDNF-like syntheticligands. Finally, new strategies are currently explored to increase the targetavailability of BDNF and expand its biological life.

Keywords:brain derived neurotrophic factor, neurodegeneration,neurotrophins, regeneration, therapy

Exp. Opin. Ther. Patents (1999)9(12):1655-1664

1. Introduction

BDNF research is second only to its neurotrophin relative, nerve growthfactor (NGF). It is now accepted that the pluripotent BDNF and its highaffinity receptor trkB are widely distributed both in the developing andmature nervous system. For this review, we read the description of BDNF(but not exclusively) oriented patents submitted between 1996 and the firsthalf of 1999 and attempted to put them in the context of the abundant litera-ture on the subject (over 250 Medline references in the first six months of1999!). We believe that neurotrophin research is, arguably, the hottest areain neuroscience today and BDNF will probably be the subject of a signifi-cant number of discoveries about mechanisms of neurodegeneration andpotential therapeutic interventions.

Our review will focus on the major directions of research and principles oftherapeutic intervention rather than an exhaustive account of progress inthe field. For this latter purpose, the reader will be directed to someexcellent reviews. The majority of bibliography is relevant to the principlesdiscussed in the patents reviewed and may not always be the ultimatereport on the topic in general. Although we tried to just summarise thepatents or scientific reports outlined in this review and then present ouropinion at the end, we sometimes felt that it would be pertinent to comment

16551999 © Ashley Publications Ltd. ISSN 1354-3776

Review

1. Introduction

1.1 The almost good news

1.2 The good news

2. BDNF: the basics

2.1 BDNF in human braindegeneration

2.2 New functions attributedto BDNF

2.3 Clinical targets

3. Recent technicaldevelopments

3.1 New experimental models

3.2 New methods to analyseBDNF and trkB activities

4. Designing BDNF basedtherapies

4.1 In vivo delivery

4.2 Bioengineering andbioavailability

4.3 Natural resources

5. Expert opinion

5.1 BDNF activities

5.2 From the researchlaboratory to the clinic

5.3 New therapeutic designs

Acknowledgements

Bibliography

Patents

http://www.ashley-pub.com

Expert Opinion on Therapeutic Patents

on the potential of specific patents in the context ofthat particular subheading and the literature cited.

1.1 The almost good news

A plethora of patents have been submitted in theincreasingly competitive field of neuroregenerativetherapy claiming that they will ‘cure’ Parkinson’sdisease (PD), Alzheimer’s disease (AD), amyotrophiclateral sclerosis (ALS) and Huntington’s disease (HD),often all at once! Of course, this is good news. Still,many patents tend to propose rather genericapproaches targeting in vivo delivery and increasedpharmacologic effects, without a clear outline of whythese treatments are considered ‘novel’ or superior tothe multitude of previously published reports.

We believe that there is still insufficient informationregarding the control of neurotrophin gene expres-sion, alternative pathways of cell signalling and,perhaps most importantly, the normal and pathologictransport of neurotrophic factors in the post-natalnervous system. In-depth knowledge of these topicsis essential for the design of scientific neurotrophinbased therapy. Furthermore, the effects of BDNF onother non-neuronal brain cells (e.g., astrocytes ormicroglia) are largely unexplored. It is potentiallydangerous to assume that BDNF will show synergisticor protective effects for all neural cells or environ-ments. Fortunately, as discussed below, significantinroads have been made for the past three years andthere is good reason for hope.

1.2The good news

Increased understanding of neurotrophin receptorbinding and signal transduction following trk and p75dimerisation and activation has lead to a series ofdevelopments in designing experimental models totest novel trophic treatments. An interesting develop-ment that has opened new directions of research is themodel of anterograde transport of BDNF and otherneurotrophins. This observation raises the possibilitythat the traditional trophic function of BDNF,described in the developing brain, may be only one ofthe many roles that it can play in the mature nervoussystem.

Another intriguing finding in the neurotrophic fieldhas been reported by investigators studying thefunctions of the low affinity receptor p75, common forall neurotrophins. When the ligand is NGF, it seemsthat p75 may mediate either a trophic or a deathsignalling function, depending on the availability of

the high affinity co-receptor trkA [1]. While itsfunctions in binding BDNF are not completelyunderstood, they seem to be exclusively trophic sinceno cell death related activities have yet been reported.Furthermore, it seems that in addition to its specificactivities, BDNF may potentiate the effects of otherNTF making it a good candidate as adjuvant inneuroregenerative or protective treatments.

2. BDNF: the basics

The most frequently described activities of BDNF,similar to other neurotrophic factors, are related to itsparticipation in neuronal survival and differentiation.BDNF was found to be potent on cholinergic,dopaminergic and glutamatergic motor and sensoryneurones, both in the central and peripheral nervoussystem. Readers interested in additional currentinformation about BDNF will enjoy the recentoverview by C Ibanez [2].

BDNF has also been implicated in the development ofthe auditory apparatus [3], including spiral andvestibular neurones [4], and possibly in the growth ofretinal neurones of the eye [5]. Finally, the participa-tion of BDNF in the development of another sensorysystem was demonstrated by Ringstedt et al. [6], whoshowed that overexpressing transgenic mice do notdevelop a normal gustatory system due to disorgan-ised neuritic sprouting.

Also interesting is the conclusion that BDNF, togetherwith other neurotrophins, may play a crucial role inthe plasticity of the human brain, especially bycontrolling neuronal cell death. This aspect of the NTFbiology has been already discussed in many publica-tions and is now standard material for theneuroscience textbooks. A comprehensive review onthe topic was published by Pettman and Henderson[7] who have integrated our knowledge about NTFdevelopmental functions with various mechanisms ofneuronal cell death. They recognise that there is agreat deal of information on the role of neurotrophinsin rapid and massive remodelling of the neuronalcytoarchitecture and connectivity in the developingand neonatal brain, but relatively little information onthe role of NTF in the adult brain. Neurones areproduced in excess during development but then theyare eliminated mostly through deprivation of NTFsupport. Many investigators believe now that in themature, terminally differentiated brain, NTF may havedifferent functions, one of them being to trigger

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1656 Brain derived neurotrophic factor and neurodegeneration

programmed cell death through less conventionaltypes of receptor signalling.

2.1 BDNF in human brain degeneration

In a comprehensive study describing the topographyof BDNF and its receptor in the human brain, Mufsonet al. showed that BDNF and trkB are widely distrib-uted, predominantly but not exclusively, on neurones[8]. Unfortunately, the baseline in the ‘normal’ humanbrain is still poorly defined and changes due tovarious pathologic conditions are difficult to interpret.

While there are few reports about BDNF reactivity inacute brain injury (e.g., cerebral ischaemia in children[9]), the majority of the reports are concerned with thedistribution of NTF and trk gene expression andproteins in chronic brain degeneration. For example,in AD, Murer et al. have shown that BDNF is oftenincreased in areas of pathology [10]. We have alsoreported an abnormal distribution in neuritic plaques[11], but our interpretation is still unclear. Others haveshown that BDNF is actually reduced in thehippocampus and temporal cortex of AD patients,both at the mRNA and protein level suggesting arationale for BDNF based therapies [12,13]. Along thesame lines, other studies show that with age, theexpression of trkB in rat brains decreases more thanBDNF [14]. Another attempt to correlate decreasedlevels of BDNF with senescent brain degeneration,like in AD, was made by Katoh-Semba et al., whofound decreased levels in the cortex of aged rats butincreased in the dentate gyrus; this furtherconfounded the interpretation of similar studies [15].

BDNF has also been proposed to have an autocrineeffect on dopaminergic neurones that expressabundant trkB [16] and if these results are confirmedin humans, new hypotheses may be formulated aboutthe mechanisms of disease in PD. Supporting thistheory is a study by Parain et al. who showed reducedBDNF protein in the substantia nigra of PD patients[17]. In AIDS dementia we have first described anabnormal distribution of BDNF in infiltratingmacrophages in HIV encephalitis accompanied byupregulation of trkB on surrounding reactiveastrocytes [18]. While the interpretation of theseresults is still speculative, perhaps questioning its(compensatory?) neurotrophic benefit, it has becomeincreasingly clear that BDNF is emerging as animportant player in a large variety of neurodegenera-tive diseases.

2.2New functions attributed to BDNF

It is believed that the neurotrophin family shares asignificant number of biological features, at least interms of gene expression and translation. While thismay be largely true, there is increasing evidence thatthere are also significant differences among neurotro-phins, beginning with post-translational processing.Mowla et al. have recently proposed an interestingconcept with potential pharmacotherapeutic implica-tions [19]. They have shown that, in contrast to NGFreleased through the constitutive secretory pathway,BDNF appears to be produced in the regulatedpathway of neurones and released in an activitydependent manner. For example, in hippocampalneurones in vitro, NTF sorting (NGF vs. BDNF) in thetrans-Golgi network was dependent on the variableefficiency of precursor cleavage by the endoproteasefurin.

As mentioned above, an intriguing new discovery isthe ability of BDNF to be transported in an antero-grade fashion. For example, it was shown that BDNFproduced by dorsal interneurones in developmentstimulates the proliferation and differentiation ofmotoneurone progenitors after anterograde transport[20]. A similar mechanism of in vivo targeting mayoccur in the post-natal nervous system where Michaelet al. have shown that BDNF can be transported fromperipheral neurones to the dorsal root ganglia [21].Another piece of evidence comes from the studies byKokaia et al. who have shown that BDNF levelsincrease significantly in a rat model of focal ischaemiabut then decrease rapidly suggesting an anterogradetransport [22]. Finally, additional supporting data forthe anterograde transport of BDNF in the adult CNSwere reported by Conner et al. [23] who demonstratedthat its distribution parallels axonal flow, includingstorage in terminals within the target.

A potentially important implication of the anterogradetransport of BDNF is its participation in synaptictransmission. This theory is supported by datasuggesting that enhancement of long-term transmis-sion is manifested through synapse consolidationrather than neuronal growth. Also, in an in vitroexplant model using hippocampal slice cultures,Frerking et al. showed that BDNF may enhancetransmission in CA1 neurones by decreasing thepost-synaptic inhibition through a pre-synapticmechanism [24]. There is evidence that at thepre-synaptic level, BDNF potentiation is mediated bycAMP [25]. In addition, BDNF is also reported to be

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able to mediate agrin-induced post-synaptic differen-tiation [26].

2.3Clinical targets

AD is probably the best-described chronic neurode-generative disease. Although intense efforts havebeen made to understand the underlying mechanismsof neuronal atrophy, there are no conclusive findingsto indicate a clear direction of research in designingneurotrophic or protective therapies. In fact, thefrustration of AD researchers is symptomatic for theentire field of neurodegeneration research. It hasbecome increasingly evident that the translation fromexperimental models to clinical trials is often difficultor impossible when one attempts to use trophicfactors as therapeutic agents. Hefti, in a position paperdiscussing the development of NTF therapy for AD,has summarised very well the debate and concernsabout various neurotrophin based preclinical studiesand the risk to overestimate the clinical benefits [27].

While the rationale for designing NTF based therapiesin neurodegenerative diseases still presents a seriousconceptual challenge, in the field of neuro-oncologythe issues seem to be better defined and more attrac-tive to biotechnology. For example, Worcester havedeveloped a methodology that may be useful intreating tumours. They claim that vectors containingnucleotide sequences of a receptor for differentiation,like BDNF, may lead to reduction of tumourgenicity[101].

As an ever expanding target for NTF treatments, spinalcord injuries have been proposed to show significantclinical benefits. For example, intrathecally infusedBDNF at the site of spinal cord injury in rats showed apositive but transient effect on local reflexes [28]. Themost dramatic impact of BDNF occurred in fullytransected spinal cords. When these chronic infusionswere stopped, the behavioural effects disappeared.BDNF was also shown to stimulate sprouting ofcholinergic fibres at the injury site, but did not effectserotonergic fibres or total axon density. Furtherdownstream, NTF, including BDNF, have been testedfor their potential therapeutic benefit in peripheralneuropathies. Unfortunately, (pre)clinical results,discussed by Apfel et al. in an excellent review, seemto be inconclusive [29].

Another area of research less explored vis-à-visneurotrophic factor dysfunction is the field of psychi-atric disorders. For example, in a review of the topic,Altar has suggested that BDNF and other

neurotrophins could eventually be employed indesigning new antidepressant therapies [30].

A very prolific area of applications for NTF treatmentsis neurotransplantation of stem and progenitor cells.In a recent review, Svendsen discusses the potential ofthese therapies based on expanding in vitro thepopulation of neuronal precursor cells [31]. Notsurprisingly, NTF are assumed to be key factors in cellproliferation and differentiation pre-implantation. Forexample, BDNF was shown to increase the number ofdopaminergic neurones in cell aggregates grown forfuture transplantation in PD patients [32]. In vivo, thegrafts could also benefit from NTF treatments. In astudy by Broude et al., it was shown that in spinal cordtransplants, the addition of BDNF increased axonaloutgrowth of axotomised neurones [33].

3. Recent technical developments

A major difficulty in neurotrophic factor research isdefining the benchmark, especially the experimentalconditions employed by various investigators.Compared to other areas of biomedical research, thisfield is typically characterised by an unusually highnumber of variables associated with the particularneuroglial environment studied: in vitro versus invivo, animal versus human, mixed neuroglial versus‘purified’ neuronal cultures, developmental stage anddifferentiation, central versus peripheral nervoussystem, neurotrophic versus neuroprotective andmany others. Still, there is now a consensus that theconfusion may begin to dissipate due to the effort ofmany laboratories to report better characterised, moredetailed and more easily reproducible experimentalsystems for measuring the effects of standardised NTFtreatments.

3.1New experimental models

Historically, the common systems to measure theneuroprotective effects of NTF were based on in vitroacute neurotoxicity followed (or not) by massiveneuronal death. While relatively easy to quantify,these types of cell survival assays sometimes tended tomeasure changes in a non-physiologic neuroglialenvironment thus making the clinical utility of theinformation obtained rather limited.

Today, a frequently discussed type of neuronal injuryis mediated by oxidative stress. As a typical example, arecent study showed that BDNF can rescue cerebellargranule neurones from oxidative stress-mediated

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1658 Brain derived neurotrophic factor and neurodegeneration

apoptotic death [34]. Because of their relative ease ofuse and potential to identify immediate clinicalapplications, the ‘neuroprotective’ experimentscontinue to be the most popular in many researchlaboratories. Nevertheless, a rapidly emerging field isbased on in vitro manipulation (i.e., expansion anddifferentiation) of neuronal progenitor cells with thegoal to use them in cell transplantation and in vivoneuroregenerative therapies.

The examples discussed here are representative of theavalanche of publications reporting, most often,similar data regarding the use of neurotrophic factorsto promote cell growth and differentiation. Forexample, endothelial growth factor (EGF)-treatedmurine cultures can be induced to differentiate intoneurones by withdrawal of EGF and supplementationwith BDNF (an increase of ~ 35% over controls).BDNF treatment results in more multipolar neuronesthan NGF treated cultures [35]. In a related system,Neuralstem Biopharm. has submitted a patentclaiming that EGF is significantly more potent thanbasic fibroblast growth factor (bFGF) as a mitogen forneuronal progenitor (stem, multipotential?) cellsisolated from the embryonic rat brain [102]. Anothergeneric proposal to enhance the proliferation anddifferentiation of neuronal progenitor cells wassubmitted by Acorda Therapeutics [103] proposing touse neurotrophins delivered by various methods invitro and in vivo. These strategies can be easilyexpanded and, in a variation on the same theme, Raoet al. have recently claimed that combining theproliferative capacity of neural blast cells and thesupportive functions of various neurotrophins couldbe a potential treatment in spinal cord regeneration[104].

An increasing number of investigators now tend toagree on some broad principles of neurotrophic factortreatments targeting the expansion of neuronalprogenitor cells. In a representative patent, investiga-tors (Weis and Reynolds) from Neurospheres describethe strategy to grow mammalian neural tissuecontaining multipotent cells [105]. The method isbased on single cell dissociation followed by prolif-eration through EGF and bFGF treatments. Neuroglialcells, especially neurones, are then differentiatedusing BDNF treatments. The use of BDNF in vitro is ahighly efficient means to support the expansion,survival and differentiation of foetal neurones. Thisobservation has been reported by several laborato-ries, including ours [36].

3.2New methods to analyse BDNF and trkBactivities

The traditional methodologies used to confirm thespecificity of various NTF activities are basedprimarily on using blocking antibodies. This oftenproved to be a difficult task, not only because thespecificity of many currently available antibodies isstill disputed. In this direction, Boehringer Mannheimhas developed a new methodology of generating highaffinity antibodies against human BDNF by using animmunisation protocol based on sequential inocula-tion of the host with piscine and human BDNF [106].

Antibodies can also be used to analyse the specificactivity of neurotrophin receptors like trkB. Sugen hasreported the development of a rather complextechnology for evaluating these activities [107].Chimeric trk receptors were produced in neural cellsvia transfection with an adenovirus and theirfunctions monitored by ELISA using a c-RET reportersystem. New activities related to the BDNF highaffinity receptor were reported by a group atSumitomo Electric [108] who, after screening a humanbrain cDNA library, have isolated the novel brainspecific factor FC99 that may be activated by signal-ling mediated through trkB.

While the assays and experimental developmentsdescribed above may enhance our ability to dissecteffects specific to BDNF, the reality is that the readoutin the majority of the current in vitro and in vivoassays are still based on generic neuronal survival anddifferentiation. We believe that an important goal, atleast for now, should be to define the differentialpotency of various trophic factors to specifically affectneuronal versus glial growth. This sounds like a ratherobvious concept but unfortunately it is too oftenblurred by vague terminology such as ‘neuroglialtrophic activities’.

4. Designing BDNF based therapies

In a typical recent study, BDNF was shown to berapidly transported across the blood-brain barrier intothe brain parenchyma after injection into the jugularvein of mice, thus suggesting a saturable transportsystem [37]. BDNF was stable and cleared from thebrain into the blood by normal reabsorption from theCSF. Still, the question of the ability to administerphysiologic or therapeutic amounts of BDNF by thisroute was not addressed in the study, nor was theeffectiveness of this procedure for regeneration

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examined. Most likely these results were achieved byavoiding BDNF clearance by the liver and supersatu-rating the truncated TrkB receptors of ependymalcells and astrocytes of the blood-brain barrier. Whilethis study is representative of many others, the clinicalimplications are unclear and many in vivo obstaclesand limitations are still to be resolved.

At a time when our basic science knowledge aboutBDNF has grown rapidly, clinical trials are seriouslylagging behind and the results are controversial at thebest. One reason for this discrepancy may lay in therather unreliable methods used for in vivo delivery. Inthis direction, much effort is invested today inimproving the clinical benefit of BDNF treatmentsthrough biotechnical breakthroughs leading tosuperior bioavailability.

4.1 In vivo delivery

As mentioned above, many investigators are still usingdirect, and rather empiric, routes of in vivo injection invarious experimental models. Furthermore, recentreports suggest that the majority of the current clinicaltrials based on intra-CNS injections are disappointing,but, for obvious reasons, some investigators disputethe preliminary conclusions. Dittrich et al. claim thatin ALS, injection of BDNF may be of clinical benefit,circumventing the previous attempts at NTF injectionsin the nervous system [38]. In another disease (PD),using a different factor (GDNF), Kordower et al.showed no clinical improvement in a patient whoreceived intraventricular injections [39].

A new approach to address the problems discussedabove was proposed by a group from Amgen whohave reported that in a rat model of kindling inducedepileptogenesis, significant changes in seizure stagesmay be achieved through intraventricular injections ofrecombinant BDNF [109]. A patent from Cytothera-peutics claims to report a delivery system, throughdirect injections of BDNF or implantation of polymercapsules, that may alleviate the previously reportedside effects in humans [110]. Clinical trials based onthese latter methodologies are under way but thepreliminary data are not encouraging. A potentiallysignificant advance in the delivery of pharmacologi-cally active compounds may result from improvedformulation and packaging of new BDNF-likemolecules.

Liposomes have become one of the trendiestapproaches to in vivo delivery. For example, Hayes etal. have reported that liposome mediated transfection

of BDNF genes, both in vitro and in vivo, may result inimproved neuronal survival [111]. A cationic liposometransfer of cytomegalovirus (CMV) promoted BDNFcDNA was achieved by stereotaxically injecting al iposome/BDNF cDNA complex into thehippocampus of rats following a cortical impactinjury. BDNF mRNA was transiently expressed for 6days. However, no data suggesting the spread ofBDNF outside of the injection site were shown [40].Additional literature suggests that this may be afeasible gene delivery system but no convincingevidence about its superiority compared to othermethodologies has yet been presented.

The most promising gene delivery system and per sein vivo induction of therapeutic BDNF expression isbased on viral vectors. The field of viral vector therapyin neuroprotection and regeneration was pioneeredby investigators studying the benefits of NGF and,more recently GDNF, in experimental models of ADand PD. For example, Bohn et al. [112] have shownthat adenoviral vectors transporting neurotrophicfactors in the brain of animals challenged with variousneurotoxins may have a protective role. The advent ofincreasingly efficient and long-term reliableplatforms, like the lentiviral vectors, has brought thisfield to the centre of therapeutic research in neurode-generative diseases. For instance, a recent reportsuggests that in vivo infection with a viral vector (AAV)carrying the BDNF gene can have long-term effects oncholinergic neurones [41]. The interest in viral vectormediated gene therapy has already surpassed celltransplantation and direct in vivo delivery ofneurotrophic protein compounds.

4.2Bioengineering and bioavailability

Under the umbrella of bioavailability, several areasare included, all of them potential targets for signifi-cant technological developments. The first andusually critical limiting step in controlling theavailability of BDNF (and many other proteins)administered systemically is their ability to cross theblood-brain barrier. By conjugating BDNF to a watersoluble polymer (e.g., Mono MPEG), attached to theN-terminal α amino group, a group from Amgenclaims to have overcome this obstacle [113].

Molecularly reformulated BDNF designed to reducehepatic uptake while avoiding interference with thetrkB binding site while linking BDNF to the OX26monoclonal antibody, which undergoes receptormediated transport at the blood-brain barrier, was

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1660 Brain derived neurotrophic factor and neurodegeneration

recently reported by Wu and Partridge. The BDNF/OX26 complex crossed the blood-brain barrier in theforebrain of ischaemic rats. No data relating to thephysiological effects of this treatment are available yet[42]. A complementary approach, to increase both thecirculating life and absorption of neurotrophins, wasdeveloped by another group at Amgen who hasproduced modified positively charged BDNFmolecules with a reduced isoelectric point [114].Tanaka and Kumano from Regeneron havedeveloped a new, reportedly more stable, formulationof BDNF based on a complex including manitol and adetergent like Tween 80 [115]. It seems that thepresence of Tween 80 is critical for preventing thepolymerisation and denaturation associated withstorage of BDNF.

While the compounds described above are largelybased on alterations of the natural neurotrophinmolecules, several groups have worked ondeveloping molecules with neurotrophic activitybased on a common neurotrophin backbone andreceptor specific activity. One of the original reportsabout ‘pantrophic neurotrophins’ was presented byGenentech and claimed a series of novel sequenceswith various specific domains [116]. In the same trend,a group from Max Plank has designed a new nucleicacid sequence coding for ‘unnaturally occurring’polypeptides with neurotrophin activity [117]. Thesecompounds have an extended N-terminus (versus thenatural shorter one) that results in a more stableprecursor in vivo. The authors claim that the novelBDNF peptides may form dimers with up to one logsuperior biological activity.

An interesting advance in the field of neurotrophicfactor based therapies is the development of smallmolecule ligands and antibodies to neurotrophinreceptors. The designer compounds can be synthe-sised to develop either agonistic or blocking activities.In a patent from McGill, the authors (Saragovi et al.)describe the significant in vivo potency of these novelligands binding to both the high and low affinityneurotrophin receptors [118]. The patent is based onseveral previous reports about the biological proper-ties of a complex between NGF and a monoclonalantibody binding to it. This complex induces fastinternalisation of ligand and TrkA and exhibits theneurotrophic but not neuritogenic actions of normalNGF [43]. If clinical trials will confirm the original invivo observations in experimental models, this maybecome one of the most promising therapeutic strate-gies. The small molecules mimicking natural

neurotrophins are characterised by superiorbioavailability and less complicated in vivo delivery.

4.3 Natural resources

Before attempting to use a neurotrophin basedmolecular pharmacologic approach to promoteregeneration, one may consider to exploit somealternative natural ways of in vivo delivery. Forexample, Batchelor et al. have shown that in striatalinjury, microglia can produce BDNF and induce THfibre sprouting [44]. Based on similar observations,strategies were developed by Lazarov-Spiegler et al.[45] who have reported that mononuclear phagocytes,after in vitro manipulation, including addition ofanti-inflammatory agents and especially neurotrophicfactors like BDNF, may promote neuroregenerationafter injection in vivo at the site of injury [119,120].

The observation that activated peripheral bloodmononuclear cells may produce BDNF [46] isintriguing and may have significant therapeuticimplications that need to be further explored. Ourgroup has reported previously that human monocytederived macrophages and microglia can be a signifi-cant source of neurotrophins in vitro, especially afteractivation. Furthermore, we have also found thatproduction of BDNF in vivo may become aberrant andpossibly mediate neurodegeneration [18].

5. Expert opinion

5.1 BDNF activities

We believe that BDNF is now the leading candidate tobecome a universal therapeutic agent in neuroregen-erative treatments and its potential as a drug will relyincreasingly on a better understanding of receptorfunctions in the human post-natal brain. A word ofcaution: the effects of BDNF on non-neuronal braincells are still insufficiently explored and it would bepremature to assume that all of them are beneficial.

One of the more intriguing discoveries is that BDNFcan be transported in an anterograde fashion. Animportant implication of this kind of flow is theparticipation of BDNF in synaptic transmission. Theseobservations have now opened some interestingareas of research with the potential to contribute tothe design of new neuroprotective therapies. Anotherincreasingly prolific area of research on which NTFstudies may have a major impact is the neurodegen-eration mediated by oxidative stress.

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5.2 From the research laboratory to the clinic

A major obstacle in comparing the results fromvarious experimental models using neurotrophicfactor treatments is the high number of variablesassociated with any particular neuroglial environmentstudied. As discussed earlier, we believe it is critical todissect out the neuronal specific versus glial effects ofpluripotent factors like BDNF and abandon thegeneric conclusions like ‘neuroglial trophic activities’.

Furthermore, it has proved to be extremely difficult toconfirm in clinical trials the results from experimentalmodels using neurotrophins as therapeutic agents.This is especially true in neurodegenerative diseaseslike AD or ALS. At the present time, the mostpromising field of NTF use is transplantation of neuralcells. The majority of investigators now agree onseveral basic principles of neurotrophic factortreatments that promote the expansion, survival anddifferentiation of neural progenitor cells.

5.3 New therapeutic designs

The majority of the recent reviews suggest that thecurrent clinical trials did not yet fulfil their originalpromises. Fortunately, the recent pharmacologicaladvances are promising and we believe that thescepticism about NTF, including BDNF, use in clinicaltreatments will soon diminish. Significant advances inthe in vivo bioavailability of BDNF may result fromimproved formulation to increase its penetrationacross the blood-brain barrier and prolong its lifewithin the CNS. Another promising approach is basedon viral vectors, especially adeno- and lentiviruses.

Finally, a very encouraging advance is the develop-ment of synthetic, small molecule ligands toneurotrophin receptors. The designer compoundscan be either agonists or antagonists and their efficacyis significantly higher compared to the natural ligands.These small molecules mimicking natural neurotro-phins are also characterised by a long shelf-life, lesscomplicated in vivo delivery and superior availabilityto the target. If clinical trials will confirm the originalin vivo observations in experimental models, this maybecome a very promising therapeutic strategy.

Acknowledgements

The authors would like to thank Dr Andrew Larnerand Dr Jeffrey Vaught for their discussion andcomments.

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Cristian L Achim† & Michael G White†Author for correspondenceUniversity of Pittsburgh School of Medicine, BST-S406, Pathology,200 Lothrop St., Pittsburgh, PA 15213, USAE-mail: [email protected]

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1664 Brain derived neurotrophic factor and neurodegeneration