novel molecular therapeutics in parkinson's disease

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11Novel molecular therapeuticsin Parkinson’s diseaseSusana Goncalves∗, Hugo Vicente Miranda∗ and Tiago F. Outeiro

11.1 Parkinson’s disease etiology and pathogenesis

Parkinson’s disease (PD) is the second most common neurodegenerative disease affecting2% of the world population over the age of 65. The most common clinical symptomsinclude muscle rigidity, resting tremor, bradykinesia and postural instability as a result ofthe loss of dopaminergic neurons in the substantia nigra pars compacta. The survivingneurons often show protein inclusions, known as Lewy bodies (LBs) and Lewy neurites,which are the pathological hallmarks of PD. Misfolded and aggregated a-syn is the mainprotein component of LBs, which can be found in both sporadic and familial forms of PD(Spillantini et al., 1997).

Mutations in the SNCA gene, which encodes for a-syn, as well as duplication and tripli-cation of the gene, are associated with familial forms of PD. The precise structure of a-synis not known. Nevertheless, it is believed to display an intrinsically unfolded structure thatmay shift to a partially folded �-helical conformation upon interaction with membranes(Zhu and Fink, 2003). Although the physiological role of a-syn is poorly understood, itis thought to be associated with synaptic function and plasticity, cell differentiation andvesicular trafficking (Klein and Lohmann-Hedrich, 2007).

In LBs the majority of a-syn protein is phosphorylated (Fujiwara et al., 2002). Otherdescribed post-translational modifications of a-syn include oxidation, ubiquitylation, ni-tration, sumoylation and glycation. However, the exact role of post-translational modifica-tions in a-syn function in both physiological and pathological conditions remains unclear.

Besides a-syn mutations, several additional genetic loci were associated with familialforms of the disease (Table 11.1). For example, the PARK2 loci encodes for Parkin,an E3 ubiquitin ligase. Parkin protein mutations are thought to result in insufficient

* These authors contributed equally to the chapter.

Molecular and Cellular Therapeutics, First Edition. Edited by David Whitehouse and Ralph Rapley.© 2012 John Wiley & Sons, Ltd. Published 2012 by John Wiley & Sons, Ltd.


246 CH11 NOVEL MOLECULAR THERAPEUTICS IN PARKINSON’S DISEASE

Table 11.1 Genetic loci linked to Parkinson’s disease*

Mutation Gene Locus Lewy bodies Onset/age Inheritance

Park 1 a-syn 4q21 yes 40s ADPark 2 Parkin 6q25 no 20s ARPark 3 ? 2p13 yes 60s ADPark 4 a-syn 4q21 yes 30s ADPark 5 UCH-L1 4p15 yes 50s ADPark 6 PINK1 1p35 ? 30s ARPark 7 DJ-1 1p36 ? 30s ARPark 8 LRRK2 12p ? 40s ADPark 9 ATP13A2 1p36 ? 10s ARPark 10 ? 1p32 ? 50s ?Park 11 GIGYF2 2q36-37 ? late ADPark 12 ? Xq21-q25 ? ? X-chromosomePark 13 HTRA2 2p12 ? 50s ?Park 14 PLA2G6 18q11 ? ? ARPark 15 FBXO7 22q12-q13 ? ? ARPark 16 ? 1q32 ? ? ?

*Abbreviations: AD, autosomal dominant; AR, autosomal recessive; ATP13A2, ATPase type 13A2; FBXO7,F-box protein 7; GIGYF2, GRB10 interacting GYF protein 2; HTRA2, HTRA serine peptidase 2, mitochondrial;LRRK2, leucine-rich repeat kinase 2; PINK1, PTEN-induced kinase 1; PLA2G6, phospholipase A2, group VI(cytosolic, calcium-independent); SNCA, a-syn; UCH-L1, ubiquitin carboxyl-terminal esterase L1.

protein clearance and subsequent protein accumulation and cellular damage (Shimuraet al., 2000).

Mutations in PARK5, the gene encoding ubiquitin C-terminal hydrolase L1 (UCH-L1),were identified in a single family with PD. UCH-L1 is also involved with proteasomaldegradation by hydrolysing the peptide-ubiquitin bonds and promoting ubiquitin recy-cling (Farrer, 2006).

The PARK7 gene, encodes for DJ-1, a protein implicated in antioxidative stress re-sponses, mainly through reactive oxidative species (ROS) scavenging (Ramsey andGiasson, 2008). Mutations in the gene encoding for PINK1 (PARK6), a mitochondrialprotein kinase, are thought to impair its kinase activity and contribute to disruption ofmitochondrial trafficking, ROS formation and protein aggregation (Valente et al., 2004;Weihofen et al., 2009; Liu et al., 2009). Moreover, mutant PINK1 is not able to translocateinto the mitochondria, where it should stimulate mitophagy (Nuytemans et al., 2010).

Leucine-rich repeat kinase 2 (LRRK2), was established as the most frequently mutatedPD gene (PARK8) (Nichols et al., 2007). Its role in PD is still unclear, but it displays ki-nase activity. LRRK2 mutations are frequently located in the domains involved in kinaseactivity and dimerization which may result in its impaired function.

11.1.1 Current therapies in PD

Most, if not all, currently available therapies for PD are just symptomatic. While theyimprove motor dysfunction and other clinical PD symptoms, they do not modify disease

https://www.researchgate.net/publication/7234797_Farrer_M_J_Genetics_of_Parkinson_disease_paradigm_shifts_and_future_prospects_Nat_Rev_Genet_7_306-318?el=1_x_8&enrichId=rgreq-2a84d18f-ac3a-4d19-bf28-2e2cd0a181c7&enrichSource=Y292ZXJQYWdlOzIzMzg1Mzg0MztBUzo5NzA3NTU4NDgzMTQ5NUAxNDAwMTU2MDMxNDM2

https://www.researchgate.net/publication/44633959_Genetic_etiology_of_Parkinson's_disease_associated_with_mutations_in_the_SNCA_PARK2_PINK1_PARK7_and_LRRK2_genes_a_mutation_update?el=1_x_8&enrichId=rgreq-2a84d18f-ac3a-4d19-bf28-2e2cd0a181c7&enrichSource=Y292ZXJQYWdlOzIzMzg1Mzg0MztBUzo5NzA3NTU4NDgzMTQ5NUAxNDAwMTU2MDMxNDM2


11.2 TARGETING PROTEIN QUALITY CONTROL SYSTEMS IN PD 247

progression nor prevent disease onset. These therapies include pharmacological modula-tion of the dopamine system, neurosurgery and physical therapy.

Since shortage of dopamine is one of the major deficits in the PD brain, current phar-macologic interventions are aimed either at replenishing dopamine levels in the brain orat modulating the dopamine system with specific agonists and antagonists. More specif-ically, the strategies are the immediate or controlled uptake of the stable dopamine pre-cursor levodopa and the inhibition of monoamine oxidase B (MAO-B) or catechol-O-methyltransferase (COMT), which are enzymes that catabolize dopamine (Goetz et al.,2005, Horstink et al., 2006). Levodopa and dopamine agonists are the most widelyused drugs, as they readily cross the blood-brain barrier (BBB) to exert their anti-parkinsonian effects. However, long-term usage of levodopa may result in motor com-plications (Olanow et al., 2004). MAO-B inhibitors, such as selegiline or rasagiline, arethought to be neuroprotective as they can inhibit dopamine catabolism. COMT inhibitorsalso act on the dopamine pathway by inhibiting levodopa catabolism and by extendingits half-life. For example, tolcapone and entacapone are effective in alleviating the motorimpairments, but they are associated with hepatotoxicity (Williams et al., 2010).

Surgical approaches such as deep brain stimulation (DBS) are presently used, wherea neurostimulator delivers electric stimuli to targeted brain areas which are responsiblefor motor control. This strategy constitutes an alternative treatment in patients who meetspecific criteria. A clinical trial comparing drug therapy with a combined drug therapy andDBS showed that patients of the latter group have an improved quality of life, regardingmotor impairment and dyskinesias although this is only a symptomatic treatment (Lozanoet al., 2010).

In order to develop novel therapeutic strategies for PD it is crucial to gain a detailedunderstanding of the molecular mechanisms involved in the disease. Since a-syn-inducedcytotoxicity seems to be mainly associated with its misfolding and aggregation, it is im-portant to understand how cells respond to the accumulation of these protein species.

11.2 Targeting protein quality control systems in PD

Quality control systems in the cell comprise the protein degradation (ubiquitin-proteasome and autophagy-lysosome) and protein folding (chaperones) systems.

11.2.1 Protein degradation systems

Several factors are known to contribute to the misfolding and accumulation of pro-teins. These include protein overexpression, certain post-translational modifications, mu-tations and environmental stress (temperature, heavy metals and UV). Misfolded anddamaged proteins can be targeted to the ubiquitin-proteasome system (UPS) to avoidaccumulation and subsequent potentially toxic effects on cells, or can be processed bythe autophagy-lysosome system (ALS) (Figure 11.1). The former is mainly involved inthe nuclear/cytosolic protein degradation, and the latter in the clearance of cytosolic or-ganelles and long-lived proteins.



Figure 11.1 The role of protein quality control systems in PD. After a-syn synthesis the protein maymisfold and self-associate to form dimers, oligomers and, ultimately, amyloid fibrils and inclusionbodies. Different HSPs can modulate the oligomerization equilibrium. Hsp70 is known to contributeto the correct folding of a-syn, preventing the formation of aggregated forms of the protein. Onestrategy to increase Hsp70 levels might be through the inhibition of Hsp90 binding to HSF-1 which inturn results in the transcriptional activation of chaperones. Oligomeric species of a-syn are believedto display cytotoxicity. These may be either directly targeted by Hsps or they may also be directedfor proteasomal degradation. Oligomeric and/or aggregated species that are not degraded by theproteasome may be processed by chaperone-mediated autophagy. Black upwards arrows representputative therapeutic strategy targets. Chap represents different chaperones.

When the activity of the UPS and ALS is compromised, accumulation of misfoldedproteins may occur. In PD, the presence of ubiquitin-positive LBs suggests that theUPS might be involved in their formation (Sampathu et al., 2003; Tofaris, Layfield andSpillantini, 2001). One possibility is that UPS function in PD might be compromisedleading to the accumulation of misfolded a-syn in the form of LBs. In support of thispossibility, mutations in Parkin and UCH-L1 are associated with familial PD. Parkin me-diates both classical K48-linked ubiquitylation and non-classical K63-linked ubiquityla-tion whose lack of function leads to proteasomal degradation failure and culminates in theformation of LBs. UCH-L1, in addition to its ubiquitin hydrolase activity, seems to have


11.2 TARGETING PROTEIN QUALITY CONTROL SYSTEMS IN PD 249

an ubiquitin ligase function that enhances the non-classical pathway of ubiquitylation,thereby promoting a-syn aggregation (Lim et al.; 2005, Liu et al., 2002).

Molecular chaperones such as Hsp70 and Hsp90 are responsible for the unfolding anddelivery of proteins to the proteasome. This process is facilitated by the C terminus ofHsp70-interacting protein (CHIP), a protein that binds these chaperones to Parkin. Inter-estingly, coexpression of CHIP in a cell model of PD suppresses a-syn oligomerization bydegrading oligomeric forms of a-syn. CHIP also promotes the ubiquitylation of LRRK2(Imai et al., 2003; Ko et al., 2009). Thus, CHIP may constitute a relevant therapeutictarget, possibly by modulating its levels or by enhancing its function.

When a-syn aggregates are not degradable by the UPS, autophagy plays a central role.There are three different types of autophagic processes: microautophagy, chaperone-mediated autophagy (CMA) and macroautophagy (Cuervo et al., 2004; Webb et al.,2003).

In microautophagy the non-specific targets fuse directly to the lysosome and thesesmall invaginations of the lysosome form vesicles that are processed intralysosomally.In CMA, unfolded proteins are directed by chaperones to the lysosome for degradation.Target proteins containing a specific consensus amino acid sequence are translocated intothe lysosome, by interacting with the lysosomal-associated membrane protein type 2A(LAMP-2A) through heat shock cognate protein of 70 kDa (hsc70). In macroautophagy,proteins, aggregates or other large cellular structures that are not degradable by the othermechanisms, are encapsulated in a double-membrane structure named autophagosomethat later fuses to lysosomes, where protein degradation occurs.

Several PD-associated proteins, such as a-syn, UCH-L1, LRRK2, DJ-1, Parkin andPINK1 contain a CMA recognition motif, KFERQ, suggesting that CMA dysfunctionmight be involved in the etiology of the disease. While the wild type form of a-syn is cor-rectly processed via CMA, the mutant forms A30P and A53T are not and may block thispathway (Cuervo, Wong and Martinez-Vicente, 2010). Those mutant forms of a-syn bindto LAMP-2A with a higher affinity than the wild type protein, leading to an inhibitory ef-fect of CMA activity (Cuervo et al., 2004). As a consequence, macroautophagy is inducedas a compensatory mechanism to degrade both wild type and A53T a-syn, but it is alsoassociated with increased cell death. Neurodegeneration might occur due to accumulatedinjuries after CMA failure, causing this delicate balance to shift towards cell death insteadof neuronal survival. In fact, the accumulation of a-syn pathogenic species in the cytosolblocks vesicular trafficking and promotes the accumulation of autophagosomes inside thecell. Vesicles that are not able to be recycled, suffer a progressive membrane damage, re-leasing the content into the cytoplasm and culminating in cell death (Xilouri et al., 2009).

In summary, both UPS and lysosomal degradation systems may constitute attractivetargets for rational drug design in order to improve the clearance of abnormally foldedproteins or protein aggregates in PD and in other protein misfolding disorders.

11.2.2 Protein folding systems

Chaperones are responsible for assisting and promoting the correct folding of nascentpolypeptide chains, participating in the refolding of misfolded proteins, and preventing



inappropriate interactions of misfolded or incompletely folded peptides (Hartl and Hayer-Hartl, 2002) (Figure 11.1). Since some molecular chaperones also display the abilityto disaggregate protein aggregates, it is possible that overexpression of these moleculeswould protect neurons from protein aggregation-associated degeneration.

Several heat shock proteins (HSPs) were shown to decrease a-syn toxicity both in vitroand in vivo. This effect was also observed with other misfolding-prone proteins associ-ated with other neurodegenerative diseases such as Alzheimer’s disease and Huntington’sdisease. They are able to bind to the solvent-exposed hydrophobic residues in their sub-strates and thereby facilitate their correct folding by cycles of ‘hold’ and ‘fold’, which insome cases may be ATP dependent (Hartl and Hayer-Hartl, 2002; Beissinger and Buch-ner, 1998).

In PD, Hsp27, Hsp40, Hsp70, Hsp90, CHIP, BAG5 and �B-crystallin, as well as com-ponents of the UPS, can be found in LBs. Hsp70 and Hsp90 have been the subject ofintensive investigation in the context of misfolding diseases such as PD. Hsp70 is respon-sible for the regulation of protein oligomerization and aggregation, preventing and reduc-ing the formation and toxicity of a-syn oligomers and other aggregated species in differentcell lines and animal models of PD. For example, expressing a-syn in a fly model sys-tem results in a loss of dopaminergic neurons and formation of intraneuronal inclusions.However, coexpressing a-syn with Hsp70 significantly reduces the loss of dopaminergicneurons, although LBs are still formed.

Hsp90 is increased in PD brains and correlates with the levels of insoluble a-synspecies, suggesting that a decrease in its levels might be protective in PD. It is knownthat inhibition of Hsp90 results in the activation of HSF-1, a transcription factor able toactivate genes with heat shock-inducible promoters, and consequently enhance Hsp70expression, resulting in a decrease of a-syn toxicity (Dickey et al., 2005; Fujikake et al.,2008). This can be achieved with geldanamycin, a natural product that inhibits Hsp90activity. However, geldanamycin is not an ideal molecule due to toxicity and its reducedability to cross the blood-brain barrier (BBB). Novel analogs of this compound are beingdeveloped and tested, and some show a higher affinity for Hsp90 and better capabilityto cross the BBB. For instance, 17-AAG (17-(allylamino)-17-demethoxygeldanamycin)was shown to up-regulate Hsp70 in a cell line system reducing a-syn protein levels andtoxicity (Putcha et al., 2010). This drug is now in phase II trials as an anti-tumour com-pound, although it causes hepatotoxicity. Other drugs that can inhibit Hsp90 have showngood BBB permeability and oral bioavailability and are now under phase I clinical trials(Chandarlapaty et al., 2008; Okawa et al., 2009). Besides Hsp90 inhibitors, other drugshave been shown to directly activate HSF-1, up-regulating Hsp70 expression. Arimoclo-mol, for instance, up-regulates HSF-1 and decreases motor neuron degeneration and alsothe number of protein aggregates in the spinal cord of a mouse model of amyotrophiclateral sclerosis (ALS), and is now in phase II trials for this disorder (Lanka et al., 2009;Kalmar and Greensmith, 2009). As in ALS, a neurodegenerative disease also character-ized by protein misfolding and aggregation in neurons, arimoclomol may also be usefulin PD. Another approach that could protect against a-syn toxicity and aggregation is thedirect overexpression of Hsp70 or the silencing of BAG5 in dopaminergic neurons.

In addition to molecular chaperones, some small molecules display chaperone-like ac-tivity, modulating protein stability and/or folding, and are known as chemical chaperones.


11.3 VESICULAR TRAFFICKING DEFECTS IN MODELS OF PARKINSON’S DISEASE 251

One of the most studied chemical chaperones in the context of PD is trehalose, a natu-ral disaccharide that has been shown to prevent aggregation of proteins in vitro (Singerand Lindquist, 1998). In mammalian cell lines, trehalose enhances the clearance of a-synvariants A30P and A53T, highlighting the importance of these molecules as therapeuticapproaches.

11.3 Vesicular trafficking defects in modelsof Parkinson’s disease

Trafficking processes govern the physiological homeostasis of the brain and, intraneu-ronally, they ensure the molecular dynamics needed to maintain cell survival. Vesiculartrafficking includes the export of newly synthesized proteins from the ER to the Golgiand, ultimately, to the cell surface. Moreover, it ensures the recycling of membrane re-ceptors and the lysosomal transportation for degradation (Figure 11.2).

Rab GTPases, a family of G protein-coupled receptors, are major players in thosecellular processes. This highly conserved family of proteins is composed of more than60 members in mammals (Zerial and McBride, 2001).

Through an RNAi screen in a C. elegans model of PD, based on the expression ofwild type-, A30P- or A53T-a-syn, components of the endocytic pathway were identifiedto play an important role in the worm neurotoxicity, growth and movement coordina-tion (Kuwahara et al., 2008). More specifically, a-syn accumulation was shown to impairendocytosis, ER-to-Golgi traffic and acetylcholine (Ach) release from the synapses.

Rab guanosine triphosphatase (GTPase) orthologs are the major players involvedin the ER-to-Golgi transport impairment. Overexpression of Rab1 in C. elegans,D. melanogaster and primary neuronal cultures, suppresses a-syn-induced toxicity. More-over, different studies showed that dysregulation of Rab members as Rab3a (involvedin exocytosis), Rab5 (important for endocytosis), Rab7 (implicated in the formationand fusion of late endocytic structures with lysosomes) and Rab8 (involved in trans-Golgi transport), can be involved in a-syn pathology (Dalfo et al., 2004). In addition,Rab3b overexpression in rat can rescue the neurotoxicity of 6-hydroxydopamine, a neu-rotoxin that selectively kills dopaminergic and noradrenergic neurons (Chung et al., 2009;Kuwahara et al., 2008).

SNARE proteins (involved in the fusion of vesicles) were also described to restoretrafficking processes in the neuron (Auluck, Caraveo and Lindquist, 2010). Interestingly,a-syn is believed to assist the folding of SNARE proteins, thereby modulating the releaseof synaptic neurotransmitters (Bonini and Giasson, 2005).

In PD, a consequence of vesicular transport impairment is the functional deficit ofthe nigrostriatal dopamine (DA) system. DA, through a decrease of the vesicular neuro-transmitter uptake, is stalled in the ER-Golgi compartments. In this case, DA is rapidlyoxidized to generate ROS, contributing for cell damage and death.

Altogether, these findings suggest that a-syn aggregation can interfere with the cellulartrafficking and, therefore, modulating vesicular trafficking function might constitute avalid therapeutic approach.



Figure 11.2 Trafficking dynamics in the neuron. Rab proteins are widely spread in the cytosol andmodulate the transport of newly-synthetized proteins, from the ER-to-Golgi, and from the trans-Golgito the plasma membrane, where Rab1, Rab8 and Rab3a appear to have an important role in exocytosis.In endocytosis processes, Rab5 seems to be involved in vesicle formation from the plasma membrane,where the external solutes are internalized into the early endosomal compartments. In the processof fusion between those compartments, SNARE proteins were shown to have an important role. Rab7,9 and 27 are involved in the formation of late endosomes and lysosomes. In the synapse, Rab3bwas identified to modulate the release of dopamine vesicles positively. In PD, an increase in a-synconcentration may impair the correct storage and delivery of dopamine metabolite, DOPAC, in thosevesicles.


11.4 A-SYN POST-TRANSLATIONAL MODIFICATIONS 253

11.4 a-syn post-translational modifications

Post-translational modifications (PTMs) are known to modulate protein conformationalchanges and function (Figure 11.3). For example, the activation of some proteins dependson phosphorylation or methylation, and their degradation is regulated by ubiquitylation.Thus, if the normal PTMs are altered, pathological conditions may arise. Therefore, it isof great importance to investigate the physiological role of PTMs of the major players inPD, namely in the context of a-syn.

11.4.1 Protein phosphorylation in PD

There is currently no consensus regarding the role of phosphorylation on a-syn toxi-city and aggregation. It is still unclear whether phosphorylation is either a trigger ora late event in a-syn oligomerization and whether modulating the activity of kinases/phosphatases can increase or decrease a-syn oligomerization and toxicity (Fig-ure 11.3(a)). Several kinases were shown to phosphorylate a-syn at Ser-129 (Inglis et al.,2009; Krantz et al., 1997; Pronin et al., 2000). Other sites, such as Ser-87 and Tyr-125,are now emerging as targets for phosphorylation and demand further investigation. Re-cently, it was observed that Tyr-125 phosphorylation decreases upon ageing and is absentin the brains of patients with dementia with Lewy bodies (Chen et al., 2009). These datasuggest that phosphorylation on Tyr-125 may be beneficial. If this hypothesis is correct,gene therapy or pharmacological interventions to modulate a-syn phosphorylation mightconstitute valid therapeutic strategies for PD.

11.4.2 Protein nitration and nitrosylation in PD

In PD, a-syn is found to be nitrated in LBs. It was proposed that protein nitration/nitrosylation – the reaction between a nitro group and tyrosine or cysteine residues – maybe one of the oxidative mechanisms responsible for the formation of di-tyrosine crosslink-ings which contribute to a-syn oligomerization (Giasson et al., 2000; Hodara et al., 2004;Souza et al., 2000). Moreover, soluble nitrated a-syn is not efficiently processed by pro-teases, leading to partial unfolding, accumulation and fibril formation (Hodara et al.,2004). Interestingly, activated microglia is found to induce nitric oxide (NO)-dependentoxidative-stress in different cell types and consequently lead to nitration of a-syn thatultimately results in neurodegeneration (Figure 11.3(b)).

Recently, an inhibitor of inducible nitric oxide synthase (iNOS), the enzyme responsi-ble for NO synthesis, was shown to be neuroprotective, suggesting that this enzyme mightalso constitute a good therapeutic target for PD.

11.4.3 Protein sumoylation in PD

A-syn can be modified by small ubiquitin-like modifiers (SUMO) in a process known assumoylation (Figure 11.3(c)). Sumoylated a-syn is also found in LBs suggesting that



Figure 11.3 Post-translational modifications in PD. (a) The role of a-syn phosphorylation remainscontroversial. Different kinases (casein kinases, CKs, and polo-like kinases, PLKs) are involved in itsspecific phosphorylation in residue S129 and may contribute to oligomerization and aggregation. (b) a-syn can be nitrated in LBs. NO may arise from microglia activation that express iNOS de novo, resultingin high levels of NO release that can modify the a-syn present in surrounding cells. Nitrosylationmay also contribute to the impairment of a-syn clearance by the proteasome and to its accumulationand aggregation. (c) Sumoylation can block proteasomal degradation of a-syn, contributing to itsaccumulation. (d) Ubiquitylation of a-syn, a process mediated by different proteins such as chaperones,Parkin and CHIP, seems to play an important role in PD, and targets a-syn to proteasome degradation.(e) a-syn mono-ubiquitylation may contribute to increased formation of inclusion bodies. (f) Glycationof a-syn contributes to its oligomerization, cross-linking and impairs its adequate polyubiquitylationand proteasomal degradation.


11.4 A-SYN POST-TRANSLATIONAL MODIFICATIONS 255

SUMO may act as a proteasome-mediated antagonist of a-syn degradation. DifferentSUMO isoforms are expressed in humans, SUMO1, 2, 3 and also SUMO4 which ishighly homologous to SUMO3 and believed to be a SUMO3 pseudogene (Su and Li,2002; Bohren et al., 2004). SUMO recognizes a specific consensus motif and poly-SUMO chains may be formed since SUMO2 and SUMO3 contain this recognition motif(Rodriguez, Dargemont and Hay, 2001; Tatham et al., 2001). Parkin is an important playerin sumoylation since it is shown to regulate the turnover of SUMO E3 ligase RanBP2,ubiquitylating and promoting its proteasomal degradation. DJ-1 is also a target for sumoy-lation in residue K130, and mutations in this residue block its correct sumoylation. SinceDJ-1 activity may rely on its correct sumoylation, dysregulation of the SUMO pathwaymay contribute to the degeneration of oxidative stress-sensitive neurons. Interestingly,DJ-1 expression is regulated by the cell oxidation levels, whereas SUMO E1 and E2 ac-tivities are reversibly inhibited. This suggests that a combination of sumoylation in Parkinand DJ-1 pathways may play a role in PD pathogenesis, and may be targeted for thera-peutic intervention.

11.4.4 Protein ubiquitylation in PD

There is an intense debate on whether ubiquitylation is a requirement for a-syn degrada-tion by the UPS or whether it may enter the 20S proteasome system directly. Nonetheless,a-syn ubiquitylation occurs in specific residues K6, K10, K12, K21 and K23 (Andersonet al., 2006) (Figure 11.3(d)).

Recent studies show that a-syn may form oligomers independently of its ubiquitylationstatus (Beyer, 2006). Nevertheless, monoubiquitylation of a-syn by seven in absentia ho-molog protein (SIAH) increases the formation of a-syn inclusion bodies within dopamin-ergic neurons and enhances its toxicity (Rott et al., 2008). These results suggest thatmonoubiquitylation may be a triggering event in a-syn aggregation (Figure 11.3(e)).

Moreover, several mutations in genes associated with the ubiquitin-proteasome systemare described as PD associated. Thus, ubiquitylation of a-syn may be a pathological eventassociated with the formation of LBs in a process that is modulated by different geneproducts, all of which might constitute targets for intervention.

11.4.5 Protein glycation in PD

Other PTMs are known to occur in the cell, such as glycation which is a spontaneousreaction between reducing sugars and free amino-groups. Since glycation agents such asmethylglyoxal, a by-product of the glycolytic pathway, are known protein cross-linkers,glycation may contribute to the chemical cross-linking and proteolytic resistance of theprotein deposits found in the LBs (Vicente, Miranda and Outeiro, 2010) (Figure 11.3(f)).This suggests that modulating the amounts of glycation agents in neurons also regulatesthe formation of inclusion bodies. One possible strategy to interfere with glycation in-volves the regulation of the enzymes responsible for the catabolism of glycating agents(mainly the glyoxalases and aldose reductase) (Maeta et al., 2005). These enzymes are



glutathione- or NADPH-dependent, which are important compounds involved in the re-sponse to oxidative stress. Strategies aimed at increasing the levels of both glutathioneand NAPDH may be important to control oxidative stress and carbonyl stress, which mayin turn prevent the aggregation of proteins such as a-syn. Interestingly, one ageing-relatedevent in PD is the decrease in glutathione levels (Thornalley, 1998) contributing to an in-crease in the formation of advanced glycation end-products (AGE), the final products ofglycation. Besides glutathione levels, the expression of glyoxalase I in normal individualsincreases until the age of 55 and progressively declines with ageing, contributing to AGEformation (Kuhla et al., 2006). These species are specifically recognized by the receptorsfor AGE (RAGE) that trigger an inflammation and oxidative stress response via NF-�Binduction and the formation of ROS. These receptors are highly expressed in PD patientswhen compared to age-matched controls, suggesting a role in the development and/orprogression of the disease (Dalfo et al., 2005). Interestingly, a synthetic derivative of vi-tamin B1, benfothiamine, was shown to prevent AGE formation in different models. In anAlzheimer’s disease mouse model, this compound was shown to improve cognitive func-tion and reduce �-amyloid deposition and tau phosphorylation (Pan et al., 2010). Theseresults suggest that it may also be beneficial in PD, and this should be further investigated.

11.5 Sirtuins as targets in PD

Ageing is a molecular process associated with morphological, physiological and func-tional alterations in the cells which are thought to reflect the accumulation of environ-mental and genetic injuries and result in the progressive failure of the regulatory systemsthat maintain the homeostasis of cells, tissues, organs and organ systems.

In the last decade, the discovery of sirtuins, class III histone deacetylase enzymes, wasa major contribution in the field of ageing due to their connection with lifespan extension.Moreover, sirtuins are shown to regulate signalling pathways linked to neurodegenerationand inflammation.

The first sirtuin to be identified was the yeast silent information regulator 2 (SIR2), ahomologue of human SIRT1 that was able to extend yeast lifespan by 30%, by repressinggenome instability. Similar results were obtained with SIR2 orthologs in C. elegans andDrosophila (Haigis and Sinclair, 2010).

Sirtuins are highly conserved in biology, from bacteria and archea to the eukarya. Inmammals, sirtuins can be mitochondrial (SIRT3-5), nuclear (SIRT1, -6 and -7) or cyto-plasmic (SIRT2) and, therefore, have different substrates.

The most studied sirtuin is SIRT1, which overexpression has efficiently protected cor-tical neurons from mitochondrial loss induced by A53T a-syn. SIRT2 inhibition (throughRNAi or with small molecules such as AGK2) rescues a-syn toxicity in vitro and in a flymodel of PD. Pharmacological manipulation of sirtuin activity has become an attractivefield in age-related disorders. Several SIRT1 inhibitors such as splitomycin, sirtinol andEX527, as well as activators such as resveratrol, SRT1720, SRT2183 and NAM analogshave now been described, enabling sirtuins to be considered attractive targets for thera-peutic intervention (de Oliveira, Pais and Outeiro, 2010).


11.7 CROSSING THE BLOOD–BRAIN BARRIER 257

11.6 Mitochondrial dysfunction in PD

Several models of PD are based on the use of toxins (MPTP, rotenone or 6-hydroxydopamine) which are known to lead to mitochondrial dysfunction and oxida-tive stress, highlighting the relevance of these pathways in PD pathogenesis. In addition,mitochondrial dysfunction is associated with autosomal recessive forms of PD throughmutations in Parkin, PINK1 and DJ-1. DJ-1 is found in mitochondria during oxidativestress. In an oxidative environment, its association with Parkin is enhanced, suggestingthat they cooperate in the same pathway. In fact, DJ-1-deficient mice and flies presentmore propensity to accumulate ROS (Mandemakers, Morais and de Strooper, 2007).

Recently, mitochondrial dysfunction has been used as a target for direct interventionusing antioxidants. However, trials using these types of molecules, such as coenzymeQ10, did not ameliorate PD symptoms (Storch et al., 2007). Peroxisome proliferator-activated receptors (PPARs) are new attractive targets to treat mitochondrial damage andoxidative stress. They belong to a nuclear receptor superfamily involved in major bio-logical processes such as inflammation, mitochondrial function, tissue differentiation andlipid and glucose metabolism.

Pioglitazone is a PPAR-� agonist which, when administrated to mice before MPTPinjection, prevents dopaminergic neuronal loss and glial cell activation, by inhibitingthe conversion of MPTP into MPP+. Concordantly, in a rat model of PD, pioglitazoneimproved mitochondrial function, dopamine levels and neuroprotection. In vitro cellstudies with rosiglitazone, another PPAR-� agonist, protected human neuroblastomacells from acetaldehyde-induced ROS and apoptosis, through the induction of antioxidantenzymes. In in vitro models, ibuprofen and acetaminophen were also shown to impairneurotoxicity by binding to PPAR-� and PPAR-�. PPAR agonists are thus promisingtherapeutic targets, but further studies are needed to prove their safety and efficacy in PDpatients. Moreover, although PD is a multifactorial disorder, the widespread involvementof PPAR in cell biology must be carefully regarded to avoid putative severe side effects(Chaturvedi and Beal, 2008).

Poly [ADP-ribose] polymerase 1 (PARP1) is a protein involved in repairing DNA dam-age caused by oxidative stress, apoptotic cascade and neurodegeneration. ROS accumu-lation is sufficient to activate PARP1. Interestingly, MPTP was shown to activate PARP1specifically in dopaminergic neurons, suggesting the involvement of PARP1 in PD.PARP1 knockout mice are protected from MPTP neurotoxicity. Since PARP1 is known tolead to severe ATP and NAD+ depletion under hypoxia and oxidation, PARP1 inhibitorsmight be useful targets in PD. The protective effects of PARP-1 inhibitors were assessedboth in a cellular model of PD, based on the overexpression of a-syn, and in MPP+-treatedrat primary neuronal cultures. PJ34 was the most potent inhibitor, having concordant ef-fect on both models. In vivo studies are now necessary to validate these results.

11.7 Crossing the blood–brain barrier

The efficient delivery of compounds to the brain is limited by brain barriers, which sepa-rate the brain from blood and cerebral spinal fluid. The BBB is a physical and chemical



barrier that maintains the brain homeostasis through a tight regulation of substances inthe blood that effectively enter the brain (brain extracellular fluid). This barrier is com-posed by endothelial cells, astrocytes, pericytes and sporadic neuronal processes. Also,it comprises a transporter system which regulates molecular traffic across the endothe-lium, based on size, charge and chemical properties, through transferring low-densitylipoprotein receptor and insulin receptors (Neuwelt et al., 2008). Several gene deliverysystems are now available and hold potential to be used for therapeutic interventions(Figure 11.4).

11.7.1 Non-viral vector delivery systems

Liposomes are small vesicles which can be used to deliver DNA or other molecules ofinterest to the brain. The vesicle capsule should be conjugated with polyethylene glycolmolecules and a brain-specific monoclonal antibody (mAb) receptor, such as the insulinreceptor, in order to target the liposomes into the brain. This mAb acts as a ‘Trojan horse’that can pass unnoticed by the surveillance systems and is able to be taken up via theendogenous transportation system across the BBB (Figure 11.4(a)). This was previouslyused in Rhesus monkeys and in rats with the use of a luciferase reporter, where the liver,spleen, lung and brain were targeted (Zhang, Schlachetzki and Pardridge, 2003).

Lactoferrin (Lf)-modified nanoparticles (NPs) are another kind of non-viral gene vec-tor already experimented in a rotenone-induced rat model of PD. Lf binds to its receptorsand the modification with NPs enhances the BBB crossing. This system was used to de-liver glial cell-line derived neurotrophic factor gene (hGDNF) intravenously, reachingdopaminergic neurons in a non-invasive manner and resulting in a significant improve-ment in motor skills. Thus, through the use of this type of delivery system, GDNF wasshown to exert specific neurotrophic effects on dopaminergic neurons, promoting cellsurvival and differentiation (Huang et al., 2010).

11.7.2 Viral vectors-mediated strategies

Lentiviral- and adeno-associated vectors were able efficiently to transduce dopaminergicneurons with high tropism. This property holds great potential for gene therapy applica-tions, both through overexpression or suppression of key proteins involved in PD.

Lentiviral vectors are powerful tools for gene transfer to the CNS (Figure 11.4(b)). Forexample, injection of a Parkin-containing lentiviral vector in a rat model of PD signifi-cantly reduced the neuronal loss in DA neurons.

Glial cell-linederived neurotrophic factor (GDNF) has been widely considered forPD therapy by its neuroprotective and neurodegenerative effects and its enhancement ofdopaminergic function in PD animal models. Although human clinical trials with GDNFdid not result in a consistent relief of PD symptoms, the release of GDNF using a lentiviralvector in a rat model of PD protects dopaminergic neurons and reflected behavioural im-provements. The development of novel GDNF-based therapies for PD must be performedwith an accurate targeting delivery of the GDNF in the PD-affected neurons (Lindvall and


11.7 CROSSING THE BLOOD–BRAIN BARRIER 259

Figure 11.4 Delivery strategies across the blood–brain barrier. Targeting receptors on the BBB mightbe an appealing strategy to deliver therapeutic proteins from the endothelial cells to the centralnervous system. (a) Using liposomes, the DNA plasmid encoding for the therapeutic gene (TG) isencapsulated in a polyethylene glycol capsule combined with a monoclonal antibody receptor-specific,such as insulin, transferrin or ApoB receptors. This liposome undergoes receptor-mediated transcytosis,crossing the BBB and reaching the target cells (b) Using a lentiviral vector, the TG is fused to a receptor-binding domain and, using the viral replicative machinery, it multiplies in the packaging cells, fromwhere the formed viruses are released into the endothelial cells. Here, they cross the BBB by targetingbrain-specific receptors (c) As rabies viruses specifically target acetylcholine receptors, these systemsare becoming attractive ways to deliver therapeutic siRNA directly against the RNA of interest, TG-siRNA, through its fusion with a peptide derived from rabies virus glycoprotein (RVG). This representsan elegant system to specifically silence genes in neuronal cells.



Wahlberg, 2008). This can be achieved using a lentiviral vector encoding a secreted formof glucocerebrosidase fused to neuronal specific receptors. For example, a lentiviral vec-tor containing the glucocerebrosidase gene (fused to a LDLR-binding domain of ApoB)travels from the spleen and liver into neurons and astrocytes in the cerebral cortex, stria-tum and olfactory cells of intraperitoneally and intravenously injected mice (Spencer andVerma, 2007).

Adeno-associated viral vectors (AAVs), are considered to be well tolerated by cellsand do not incorporate into the host genome with high frequency, as shown in preclinicalstudies performed in PD models such as rodents and primates.

AAV9 lentiviral vectors were shown predominantly to infect astrocytes in adult miceand neurons and lower motor neurons in the neonate (Foust et al., 2009). AdenoviralAAV2/6 vectors showed a high tropism for DA neurons, being able to infect about 80%of nigral dopaminergic neurons (Schneider, Zufferey and Aebischer, 2008).

The delivery of glutamic acid decarboxylase 65 (GAD65) gene in an AAV vector in-creased GABA release in a rat PD model. This is a promising effect as the neuronaldecrease of dopamine results in reduced GABA neurotransmission (inhibitory). This canlead to a deregulated increase of glutamate (excitatory) neurotransmission which culmi-nates in the motor impairment symptoms of PD. To overcome these imbalanced excita-tory pathways in SN, therapy with GAD65 shifts predominantly excitatory responses toinhibitory ones and rescues neurons from toxicity (Kim et al., 2008).

Another interesting approach is the transvascular delivery of small interfering RNA(siRNA) into the brain, in order to silence the expression of a specific gene (Fig-ure 11.4(c)). As neurotropic viruses are able to cross the BBB and infect cells, viralglycoproteins such as rabies virus glycoprotein (RVG) are a molecular vehicle to de-liver loss-of-function particles into the neurons. A peptide derived from RVG is fusedto double-stranded RNA. Upon interaction with brain nicotinic acetylcholine receptor,it effectively delivers the RNA in neuronal cells (thalamus, striatum and cortex) ratherthan spleen or liver. This RNA is further processed and forms the single-strand siRNAwhich will suppress the expression of a specific target gene (Kumar et al., 2007). In sum-mary, the requirement for novel therapeutics is contributing to the development of novelstrategies for the efficient and specific delivery of genes or compounds into the brain.

11.8 Concluding remarks

PD integrates a group of neurodegenerative diseases associated with protein misfoldingand aggregation. Since oligomeric and prefibrillar forms of these proteins seem to displayhigher cytotoxicity than the mature aggregated forms, future therapeutic strategies musttarget these protein species. Thus, the elucidation of the molecular mechanisms involvedin protein misfolding and the associated proteotoxicity is essential for the design of noveltherapeutic strategies for neurodegenerative disorders. In PD it is important to investigatethe mechanisms by which the protein quality control systems, vesicular trafficking, post-translational modifications and mitochondrial dysfunction modulate a-syn aggregationand toxicity.


11.8 CONCLUDING REMARKS 261

Evidence from PD patients and animal models suggest that oligomeric species of a-cynare neurotoxic and the cytoplasmic inclusions may be the result of a protective mechanismto refrain the toxic intermediates. Hence molecular modifiers that can shift the oligomer-ization of a-syn, either by inhibiting the initial monomer interactions or by promoting cy-toplasmic inclusion formation, are promising drugs. However, precluding the pathogenicspecies by inducing inclusion formation must be carried out with caution since it maybe beneficial only in a short-term perspective: aggregates may physically clog cellularprocesses and sequester different important proteins, crucial for cell survival. A promis-ing therapeutic approach should then combine different molecular modifiers of proteinoligomerization.

The use of molecular and chemical chaperones will be beneficial in a first stage tostabilize the native structure of proteins, preventing their initial misfolding. Hsp70 standsout as a great therapeutic target by preventing a-syn oligomerization, whereas HSF-1 andHsp90 appear as central modulators of Hsp70 protein levels.

Since PTMs also play a role in the oligomerization and degradation of a-syn and otherPD associated proteins, the use of specific drugs able to block or promote different PTMsmay also prevent a-syn oligomerization and toxicity.

After this, the combined modulation of the proteasomic/autophagic pathways to clearthe toxic species with aggregation inhibitors, can also constitute a promising strategy tobe tested.

Genetic or chemical modifiers of vesicular trafficking may also be important regulatorsof neurotoxicity. Namely, the enhancement of dopamine storage and adequate synapticrelease with specific Rab/SNARE proteins could restore the normal function of dopamin-ergic neurons.

Re-establishing mitochondrial function is also a major therapeutic target, since severalpathways and PD-associated proteins are known to lead to mitochondrial dysfunctionand cell death. This can be achieved by enhancing PINK1 and DJ-1 functions, responsi-ble for preventing protein accumulation, malfunctioned mitochondria removal and ROSscavenging, respectively. Also, the use of specific PPARs agonists may represent a goodtherapeutic target, since they prevent dopaminergic neuronal loss. PARP1 suppressionmay also result in mitochondrial improvement in pathologic conditions, by preventingATP and NAD+ depletion.

Sirtuins, which are involved in ageing processes, may also represent a good ther-apeutic target, since SIRT1 overexpression and SIRT2 inhibition may prevent a-syntoxicity.

Therapeutics may rely on drug- and/or gene-mediated strategies. The challenge of tar-geting the molecules, genes or virus to the brain and across the BBB is the major limita-tion. However, elegant systems to circumvent this barrier are under development. Theseinclude liposomes, Lf modified NPs, lentiviral and AAvs delivery systems and also thetransvascular delivery of siRNA. By associating specific brain-recognizable decoys, asuccessful delivery might be achieved.

Importantly, the effectiveness and timeliness of the strategies presented here mightdepend on the stage of the disease and also the exact causative mechanisms, suggestingthat tailored-therapeutics must be developed.



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