a-Syn is a 140-amino-acid protein identified as a hallmark constituent of Lewy bodies present in a group of neurodegenerative diseases, including PD, multiple system atrophy (MSA), dementia with Lewy bodies (DLB), and diffuse Lewy
body disease (DLBD). Despite the implication of nuclear localization in the nomenclature, a-syn is primarily localized to presynaptic terminals in the central
nervous system (CNS) and is abundant in brain areas rich in synaptic vesicles and associated with synaptic plasticity, such as the hippocampus, cerebral cortex, and amygdala. Early studies in songbirds (Zebra finch) demonstrate a role for a-syn in synaptic plasticity and that song learning coincides with the upregulation of a-syn mRNA. The physiologic function of a-syn in normal brain is poorly understood.
a-Syn may play a role in neuronal differentiation, regulation of dopamine release, regulation of cell viability, modulation of synaptic transmission, and vesicular recycling. Additional roles in cell adhesion, development, regulation of dopamine uptake, and vesicle transport in neurons are described.
As a highly abundant neuronal protein in the mammalian brain, a-syn interacts with a number of proteins: acting as a high-affinity inhibitor of phospholipase D2, as a regulator for certain enzymes, transporters, and neurotransmitter vesicles, promoting oxidative stress, and as a regulator for the MAP kinase pathway by forming a complex with transcription factor Elk. a-Syn also plays a role in modulating the architecture of membrane lipid components by associating with lipid membranes, fatty acids, detergent micelles, lipid rafts, and lipid droplets. Metal ions such as Cu2รพ and potentially Fe2+, Al3+, Zn2+, Mg2+, Ca2+, Co2+, Fe3+, Tb3+ and Mn2+ interact with a-syn, although a-syn is not widely regarded as a traditional metalloprotein. a-Syn also displays characteristics of chaperone-like proteins and interacts with a family of ubiquitous cytoplasmic chaperones including 14-3-3 proteins, in addition to other abundant proteins like protein kinase C (PKC), the bcl-2 homologue
BAD, and extracellular regulated kinase (ERK).
The endogenous function of a-syn has not been clearly delineated through characterization of mice deficient in a-syn expression. The first reports of mice deficient in a-syn demonstrated normal synaptic architecture and brain morphology
that led to slight changes in synaptic transmission. Additional laboratories have generated a-syn–knockout mice in combination with knockout of the two other synuclein family members in mammals, b-synuclein and g-synuclein, with little
to no apparent phenotype. A reproducible phenotype for mice deficient in a-syn includes heightened resistance to the neurotoxin MPTP. MPTP, specifically
MPP+ generated by MAO-B activity, targets susceptible dopaminergic neurons and can inhibit mitochondrial complex I activity, although the importance of mitochondrial inhibition in initiating cell death remains in question. Because the exact mechanism of MPTP action in neurons is not clear, inferring a-syn function via MPTP resistance
becomes difficult. The implication that cells containing a-syn may be more susceptible to environmentally derived toxins is provocative, but mice overexpressing a-syn may not be more susceptible to MPTP, and, in some cases, are
protected against neuronal toxins like paraquat. One explanation may involve the lack of functional overlap between mouse a-syn and human a-syn in neurons.
Clear orthologues to a-syn may not exist in lower organisms and invertebrates that would serve as models for study, further hindering efforts to understand the normal function of a-syn in cells. If native a-syn function is important for pathogenesis
and that associated function is largely unknown, inserting a-syn into organisms that are normally devoid of the protein without the ability to assess whether a-syn integrates properly into the cell would produce a model system difficult
to interpret. a-Syn function may modify crucial physiologic events in mammalian neurons that necessitate high redundancy from other proteins. Conversely, a-syn may play a more generalized role as a dispensable cofactor for a number of diverse cellular pathways present in higher organisms. The lack of a clearly described role for a-syn in cells negatively affects viability as a therapeutic target, because alteration or disruption of a-syn in humans may produce unanticipated
and deleterious side effects that outweigh potential benefits.
Monday, June 15, 2009
Friday, June 12, 2009
Focus on another PD protein: Alpha -Synuclein (a-syn)
The first genetic cause for PD was described in a large Italian family that inherited early-onset PD in an autosomal dominant fashion. Subsequently, missense mutations in the a-syn gene were also identified in Greek, German, and
Spanish families, with missense mutations localized to the N-terminal half of the protein. As opposed to missense mutations in LRRK2, pathogenic missense mutations
in the a-syn gene seem confined to only a handful of PD cases worldwide. Again in contrast with LRRK2, genetic variation in the a-syn promoter and other regions of the gene appears to modify susceptibility to PD. The identification of genomic multiplications that include a-syn and are causative for PD solidifies the importance of a-syn dosage in PD. The main strength that suggests a critical involvement
for a-syn in PDdoes not necessarily lie with human genetic studies; rather, a-syn represents the major protein component of the pathologic structures that define PD associated lesions in affected regions of the brain.
Spanish families, with missense mutations localized to the N-terminal half of the protein. As opposed to missense mutations in LRRK2, pathogenic missense mutations
in the a-syn gene seem confined to only a handful of PD cases worldwide. Again in contrast with LRRK2, genetic variation in the a-syn promoter and other regions of the gene appears to modify susceptibility to PD. The identification of genomic multiplications that include a-syn and are causative for PD solidifies the importance of a-syn dosage in PD. The main strength that suggests a critical involvement
for a-syn in PDdoes not necessarily lie with human genetic studies; rather, a-syn represents the major protein component of the pathologic structures that define PD associated lesions in affected regions of the brain.
Friday, June 5, 2009
LRRK2 Kinase activity: Tergetting the kinase
The unambiguous identification of a protein included in the so-called druggable genome clearly linked with PD susceptibility provides the opportunity to exploit existing technology to move beneficial molecules expediently to the clinic.
For example, large panels of active recombinant protein kinases are now commercially available to help define the specificity of kinase inhibitors early in the development process. Protein kinase inhibitors have proven efficacy in the
treatment of human disease since the successful application of the first approved kinase inhibitor trastuzumab in cancer therapies. After the approval and successful implementation of the small-molecule imatinib kinase inhibitor spurred the
formation of many kinase inhibitor discovery programs that are now a ubiquitous part of the modern pharmaceutical industry. However, protein kinases are far from ideal targets because problems with specificity plague the safety record of
inhibitory compounds. Additional problems such as loss of sensitivity of the drug due to acquired mutations in cancer cell targets would presumably not present an issue for the treatment of PD. However, an LRRK2 inhibitor may need
to be administered for the remainder of the patient’s life, requiring
a difficult-to-achieve level of safety from a potential inhibitory compound in the more vulnerable group of older individuals with PD. In addition, compounds would have to cross the blood–brain barrier freely to target the cells of interest.
The unique biology of the LRRK2 protein presents both an opportunity for specificity and unique challenges in identifying possible LRRK2 kinase inhibitors. Classic
protein kinase inhibitors might be grouped together as ATP-competitive inhibitors and irreversible inhibitors. The ATP-binding pocket within a protein kinase is an ideal target for compounds that possess drug-like properties, although ATP-binding pockets tend to encode some of the highest sequence homology found between different protein kinases. Residues critical to the formation of the ATP-binding pocket are conserved between kinases but not necessarily near in amino acid sequence. The majority of kinases, including LRRK2, have not been described on a structural level.
Description of the structure of the LRRK2 ATP-binding pocket and comparison with known ATP-binding pockets of other protein kinases should help shed light on whether ATP competitive compounds will be feasible for an LRRK2-based therapy. The activation loop of protein kinases, almost always defined as the sequence lying between the DFG…APE canonic sequence motif, have been used as targets for small
molecules because the activation loop is critical to kinase activity. The p38 protein kinase inhibitor BIRB796 causes a switch from a ‘‘DFG-in’’ conformation to a ‘‘DFG-out’’ conformation, leading to a steric clash with the phosphate groups
of ATP. LRRK2 possess a unique activation-loop sequence ‘‘DYG,’’ distinct from the nearly ubiquitous ‘‘DFG’’ found in protein kinase activation loops. The PD-causing
G2019S mutation further disrupts this motif to ‘‘DYS,’’ and proves the importance of these particular residues for LRRK2 kinase activity. Theoretically, a small molecule might exist that possesses activity similar to that of BIRB796 by taking
advantage of the unique structure of the LRRK2 activation loop in blocking kinase activation in a highly specific manner. Likewise, a small molecule could preferentially interact with the DYS motif in patients that carry the G2019S mutation for customized therapy, in case inhibition of LRRK2 as a whole causes intolerable side effects in humans. LRRK2 protein resides as membrane-associated and
freely soluble protein in the cytosol so that LRRK2 ATP pocket binding compounds must compete with intracellular ATP concentrations to 10mM to achieve inhibition. In addition to ATP-competitive inhibitors, irreversible inhibitors also represent a viable option for a LRRK2 kinase inhibitor, but the usual concerns of specificity and safety with irreversible inhibitors limit desirability. The safety of inhibiting
LRRK2 kinase activity and potentially LRRK1 kinase activity in humans can obviously not be fully described without highly selective and potent inhibitory molecules. In limiting undesirable effects due to the loss of LRRK activity, the available target-validation data and human genetic discoveries suggest that the LRRK2 kinase may not have to be fully inactivated to provide neuroprotection in PD, because the most common pathogenic mutations induce a relatively mild upregulation (around twofold in vitro) of kinase activity. Relevant model systems will prove invaluable in
this regard.
If the relatively unique structure of the LRRK2 kinase domain suggests that specific small-molecule inhibitors might exist, the numerous potential mechanisms for disruption of LRRK2 kinase activity through nonclassic inhibition offer another level of opportunity. In vitro data suggest that disruption of GTP-binding or nucleotide exchange within the LRRK2 GTPase domain would necessarily disrupt kinase
activity. Likewise, allosteric modulators that block a necessary conformational state, such as protein dimerization, could potentially inhibit LRRK2 kinase activity in a highly specific way (78). Substrate-competitive inhibitors would rely on the discovery of robust LRRK2 kinase targets in cells. As yet, even the sites for LRRK2 autophosphorylation have not been mapped, because of the notoriously low activity
of LRRK2 in cells and in vitro. However, the numerous technical challenges that will certainly be resolved with time are dwarfed by the potential of LRRK2 as the most exciting and viable target yet identified in PD.
For example, large panels of active recombinant protein kinases are now commercially available to help define the specificity of kinase inhibitors early in the development process. Protein kinase inhibitors have proven efficacy in the
treatment of human disease since the successful application of the first approved kinase inhibitor trastuzumab in cancer therapies. After the approval and successful implementation of the small-molecule imatinib kinase inhibitor spurred the
formation of many kinase inhibitor discovery programs that are now a ubiquitous part of the modern pharmaceutical industry. However, protein kinases are far from ideal targets because problems with specificity plague the safety record of
inhibitory compounds. Additional problems such as loss of sensitivity of the drug due to acquired mutations in cancer cell targets would presumably not present an issue for the treatment of PD. However, an LRRK2 inhibitor may need
to be administered for the remainder of the patient’s life, requiring
a difficult-to-achieve level of safety from a potential inhibitory compound in the more vulnerable group of older individuals with PD. In addition, compounds would have to cross the blood–brain barrier freely to target the cells of interest.
The unique biology of the LRRK2 protein presents both an opportunity for specificity and unique challenges in identifying possible LRRK2 kinase inhibitors. Classic
protein kinase inhibitors might be grouped together as ATP-competitive inhibitors and irreversible inhibitors. The ATP-binding pocket within a protein kinase is an ideal target for compounds that possess drug-like properties, although ATP-binding pockets tend to encode some of the highest sequence homology found between different protein kinases. Residues critical to the formation of the ATP-binding pocket are conserved between kinases but not necessarily near in amino acid sequence. The majority of kinases, including LRRK2, have not been described on a structural level.
Description of the structure of the LRRK2 ATP-binding pocket and comparison with known ATP-binding pockets of other protein kinases should help shed light on whether ATP competitive compounds will be feasible for an LRRK2-based therapy. The activation loop of protein kinases, almost always defined as the sequence lying between the DFG…APE canonic sequence motif, have been used as targets for small
molecules because the activation loop is critical to kinase activity. The p38 protein kinase inhibitor BIRB796 causes a switch from a ‘‘DFG-in’’ conformation to a ‘‘DFG-out’’ conformation, leading to a steric clash with the phosphate groups
of ATP. LRRK2 possess a unique activation-loop sequence ‘‘DYG,’’ distinct from the nearly ubiquitous ‘‘DFG’’ found in protein kinase activation loops. The PD-causing
G2019S mutation further disrupts this motif to ‘‘DYS,’’ and proves the importance of these particular residues for LRRK2 kinase activity. Theoretically, a small molecule might exist that possesses activity similar to that of BIRB796 by taking
advantage of the unique structure of the LRRK2 activation loop in blocking kinase activation in a highly specific manner. Likewise, a small molecule could preferentially interact with the DYS motif in patients that carry the G2019S mutation for customized therapy, in case inhibition of LRRK2 as a whole causes intolerable side effects in humans. LRRK2 protein resides as membrane-associated and
freely soluble protein in the cytosol so that LRRK2 ATP pocket binding compounds must compete with intracellular ATP concentrations to 10mM to achieve inhibition. In addition to ATP-competitive inhibitors, irreversible inhibitors also represent a viable option for a LRRK2 kinase inhibitor, but the usual concerns of specificity and safety with irreversible inhibitors limit desirability. The safety of inhibiting
LRRK2 kinase activity and potentially LRRK1 kinase activity in humans can obviously not be fully described without highly selective and potent inhibitory molecules. In limiting undesirable effects due to the loss of LRRK activity, the available target-validation data and human genetic discoveries suggest that the LRRK2 kinase may not have to be fully inactivated to provide neuroprotection in PD, because the most common pathogenic mutations induce a relatively mild upregulation (around twofold in vitro) of kinase activity. Relevant model systems will prove invaluable in
this regard.
If the relatively unique structure of the LRRK2 kinase domain suggests that specific small-molecule inhibitors might exist, the numerous potential mechanisms for disruption of LRRK2 kinase activity through nonclassic inhibition offer another level of opportunity. In vitro data suggest that disruption of GTP-binding or nucleotide exchange within the LRRK2 GTPase domain would necessarily disrupt kinase
activity. Likewise, allosteric modulators that block a necessary conformational state, such as protein dimerization, could potentially inhibit LRRK2 kinase activity in a highly specific way (78). Substrate-competitive inhibitors would rely on the discovery of robust LRRK2 kinase targets in cells. As yet, even the sites for LRRK2 autophosphorylation have not been mapped, because of the notoriously low activity
of LRRK2 in cells and in vitro. However, the numerous technical challenges that will certainly be resolved with time are dwarfed by the potential of LRRK2 as the most exciting and viable target yet identified in PD.
Wednesday, June 3, 2009
In vivo models of LRRK2
Many laboratories interested in developing models of PD have used LRRK2 overexpression in various organisms as a way to understand how mutations in this protein might cause PD. Simultaneously, this work will help validate or exclude
LRRK2 as a potential therapeutic target. The LRRK2 gene is highly conserved through evolution, although in many invertebrates, deciding whether the orthologue more closely resembles human LRRK1 or LRRK2 becomes challenging. Overexpression of the Drosophila orthologue of the human LRRK genes (dLRRK) appears well tolerated by Drosophila cells, and loss of the orthologue results in defects in dopaminergic
neurons, although this result has not recapitulated in other strains of flies. Expression of human WT or G2019S-LRRK2 in Drosophila results in cellular toxicity in both photoreceptor cells and dopaminergic neurons. Overexpression of dLRRK containing missense mutations in areas of the protein homologous to PD-associated mutations in the human LRRK2 gene causes significant loss of TH-positive cells, whereas expression of wild-type dLRRK or dLRRK2 with mutations predicted to inactivate kinase activity does not cause similar phenotypes. The initial studies in Drosophila suggest that LRRK2, particularly LRRK2 kinase activity, is a
valid target important for neurodegeneration. The identification of PD-causative mutations in the a-syn gene more than 10 years ago led to the description of transgenic mice overexpressing human a-syn*2.5 years later. The last 10 years witnessed numerous dramatic and impressive advances in transgenic technologies. However, since LRRK2 mutations in PD cases were described, transgenic
rodents with phenotype have yet to be described in the literature some 4 years later. Problems with cloning and manipulating LRRK2 constructs in combination with downstream expression issues seem to plague the field and may prevent fundamental questions regarding LRRK2 and neurodegeneration from being addressed in a timely fashion. Once the technical issues are resolved, rodent transgenic models may provide a springboard toward identifying and validating not only LRRK2 as a target for disease but also validating potential therapies as they arise to mitigate pathogenic processes.
LRRK2 as a potential therapeutic target. The LRRK2 gene is highly conserved through evolution, although in many invertebrates, deciding whether the orthologue more closely resembles human LRRK1 or LRRK2 becomes challenging. Overexpression of the Drosophila orthologue of the human LRRK genes (dLRRK) appears well tolerated by Drosophila cells, and loss of the orthologue results in defects in dopaminergic
neurons, although this result has not recapitulated in other strains of flies. Expression of human WT or G2019S-LRRK2 in Drosophila results in cellular toxicity in both photoreceptor cells and dopaminergic neurons. Overexpression of dLRRK containing missense mutations in areas of the protein homologous to PD-associated mutations in the human LRRK2 gene causes significant loss of TH-positive cells, whereas expression of wild-type dLRRK or dLRRK2 with mutations predicted to inactivate kinase activity does not cause similar phenotypes. The initial studies in Drosophila suggest that LRRK2, particularly LRRK2 kinase activity, is a
valid target important for neurodegeneration. The identification of PD-causative mutations in the a-syn gene more than 10 years ago led to the description of transgenic mice overexpressing human a-syn*2.5 years later. The last 10 years witnessed numerous dramatic and impressive advances in transgenic technologies. However, since LRRK2 mutations in PD cases were described, transgenic
rodents with phenotype have yet to be described in the literature some 4 years later. Problems with cloning and manipulating LRRK2 constructs in combination with downstream expression issues seem to plague the field and may prevent fundamental questions regarding LRRK2 and neurodegeneration from being addressed in a timely fashion. Once the technical issues are resolved, rodent transgenic models may provide a springboard toward identifying and validating not only LRRK2 as a target for disease but also validating potential therapies as they arise to mitigate pathogenic processes.
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