Neurodegenerative Diseaseas (Parkinson's: LRRK2) & GPCRs

Reducing the expression of alpha-synuclein

2009-11-02T10:27:00.002-06:00

Human genetic studies suggest that a-syn overexpression correlates with PD, at least in the PD cases that have multiplications of the a-syn gene. Targeting a-syn expression itself may be an effective neuroprotective therapy for an a-synucleinopathy. Recent developments highlight the current focus onRNA interference (RNAi)-mediated knockdown of asyn mRNA, thus offering protection for dopaminergic neuroblastoma cells as well as in in vivo model systems. RNAi approaches continue to make strides toward broad use in the clinic, and delivery to the central nervous system will represent one of the most difficult challenges.
If a-syn expression is required for pathogenesis, targeting a-syn mRNA should provide neuroprotection or neurorestoration or both. Outside of RNAi approaches, the asyn promoter may represent a viable target to knockdown asyn expression. GATA-2 critically induces a-syn expression, and inhibitory agents blocking transcription activation may enable therapeutically beneficial lower production of asyn,
at least in those PD patients with a-syn gene multiplications. Transcriptional regulation of a-syn through NGF- and bFGF-mediated signal transduction via the MAP-ERK and PI3 kinase pathways may also serve to regulate a-syn expression
and provide neuroprotection. Both the proteasome and lysosomal degradation pathways
seem important for clearance of a-syn and a-syn–containing protein aggregates. Enhancing proteosomal function by overexpressing parkin (E3 ubiquitin ligase) reduces a-syn toxicity in model systems, including preservation of TH positive
neurons in the substantia nigra and sparing of TH positive nerve terminals in the striatum. Decreasing a-syn ubiquitination by blocking the E3 ubiquitin-ligase SIAH
reduces the amounts of amorphous aggregates formed in cells. The action of PD-associated point mutations may impair normal metabolism through chaperone-mediated autophagy. Afterward, upregulation of cathepsin D to enhance lysosomal function promotes a-syn degradation and inhibits aggregation and toxicity. A small proportion of a-syn also exists outside of the cell and in the cerebrospinal
fluid. a-Syn vaccination may be another strategy to reduce a-syn protein levels with proof of concept demonstrated in a transgenic model in which immunization reduced
aggregation and neurotoxicity.

Aggregation Inhibitors / Toxicity inhibition for alpha-synuclein

2009-10-22T08:15:00.002-05:00

Dramatically different methods and approaches have been described that can suppress a-syn aggregation and provide protection from toxicity in model systems. Blocking the formation of aggregated a-syn structures by inhibition with short synthetic peptides represents one avenue. Peptides derived from the N-terminal amino acid sequence (1 to 15) of b-synuclein display neuroprotective activity. This peptide
sequence may be an antiaggregation factor for a-syn toxicity induced by oxidative stress. When peptide fragments are derived from the a-syn protein itself, some sequences demonstrate the propensity to inhibit fibril formation and toxicity. Overall, the peptides may act as b-sheet breakers; the shortest peptide, RGAVVTGR-amide, retains the ability to inhibit a-syn aggregation. In culture systems, a cell-permeable peptide inhibitor of a-syn aggregation inhibited DNAdamage
induced by Fe2þ in neuronal cells expressing mutant human a-syn (A53T). Therapeutic delivery of b-sheet–breaking peptides to the brain of PD-affected individuals represents a monumental challenge, but small peptides may demonstrate
proof of principle in model systems that correlate a-syn aggregation
with toxicity.
b-Synuclein (a 134-amino-acid protein) also prevents a-syn aggregation in vitro and in double-transgenic mice. Acting as chaperones, b- and g-synuclein both reduce the rate of a-syn fibrillation and aggregation. Hsp70 is a potent protein chaperone and refolding complex also known to inhibit aggregation and fibril formation by preferential binding to prefibrillar species. Hsp70 likewise potently reduces a-syn self-interaction in bimolecular fluorescence complementation assays. Other chaperones
that may play critical roles in anti–a-syn aggregation include torsinA , Hsp40, and aB-crystallin.
Broad therapeutic delivery of protective proteins that target a-syn toxicity and aggregation throughout the brain requires huge advances in current gene-therapy technology, but in the meantime, proteins that protect from a-syn toxicity in relevant model systems will help delineate pathogenesis and provide
additional therapeutic targets that in turn may be amenable small-molecule modification.
Human single-chain antibody fragments (scFv) that bind asyn inhibit toxicity and formation of a-syn–positive fibrils. scFv molecules can bind specifically to an oligomeric form of a-syn and prevent aggregation and interaction with the cell membrane, thereby reducing membrane damage and pore formation. Like peptides with an affinity for b-sheets and chaperones with an affinity for unfolded or aggregated proteins, the isolated scFvs ideally bind only to the toxic oligomeric
species in the target protein while avoiding the problem of interaction with the potentially benign and abundant natively unfolded a-syn protein. Intrabody therapy, as with most recombinant protein approaches in therapeutics, introduces
an additional set of difficult technical challenges in the clinic beyond the question of target relevance.
Inflammation and microglial activation coincide with neurodegeneration in PD and in many models of the disease. Microglia inflammation inhibitors and antiinflammatory approaches are under investigation for preventing a-syn toxicity as well as to suppress neuroinflammation in PD. Suppressive action by NSAIDs on dopamine quinone
formation by interaction of a-syn with microglia and astrocytes either may arrest or effectively slow neurodegeneration. a-Syn mutations may induce a proinflammatory
phenotype in both microglia and astrocytes, indicating the involvement of cell-surface receptors for both microglia and astrocytes. Antagonists for these putative cell surface receptors (microglial and astrocyte), as well as those
for other molecules that regulate microglial activation, including MMP-3, CD40L, CCL2, and other chemokines could constitute novel targets for therapeutic intervention.
In another study in which a commercially available compound library was screened, it was found that dopamine and other catecholamines interacted with a-syn protofibrils and inhibited the fibrilization process. When antioxidants like sodium metabisulfite were added, the process was reversed, suggesting that fibril inhibition, protofibril accumulation, and monomer modification is a sequel to covalent modification
by the dopamine-derived orthoquinone. Other compounds with antioxidative properties such as flavonoid baicalein and some antibiotics like rifampicin are
also able to inhibit a-syn fibrillation in vitro and further disaggregate
preformed fibrils and soluble oligomers. PD therapeutic agents such as selegiline, dopamine, pergolide, and bromocriptine dose-dependently inhibit the formation of asyn
fibrils and also destabilize the preformed a-syn fibrils. The potency of these compounds ranks as follows: selegiline¼dopamine>pergolide>bromocriptine. In short,
small molecules exist that likely modify a-syn structure in cells. Targeted screens that elucidate molecules with drug-like properties and demonstrate efficacy in relevant model systems will ultimately test the role of aggregation and a-syn
protofibril formation in disease pathogenesis.

Therapeutic modification of alpha-synuclein

2009-10-21T15:02:00.001-05:00

In a disease that ultimately involves much of the nervous system, small molecules that provide neuroprotection and neurorestoration to PD-affected brain areas represent the
most obvious therapeutic strategy. a-Syn is the major species composing Lewy bodies. The aberrant accumulation of a-syn likely plays a major role in the neurodegeneration
and progression of PD. From the standpoint that PD manifests as an a-synucleinopathy caused by an overabundance of the protein due to gene multiplication, associated
noncoding promoter variation that upregulates a-syn expression, or protein misfolding that results in enhanced fibrillization and decreased turnover spurred by any
number of factors, multiple levels of intervention in the disease
process may exist.

In Vivo models of alpha-synuclein

2009-10-19T08:51:00.002-05:00

Animal models involving manipulation of a-syn expression have unquestionably led to a better understanding of the correlation between a-syn, neurotoxicity, aggregation, and neurodegeneration. In a transgenic nematode model,overexpression of a-syn results in increased lifespan but impairs survival and function of the eight dopamine containing cells intrinsic to the animals. MPPþ exposure induces
dopamine-neuron death and worm lethality in a-syn transgenic worms. In this model, the major cause of MPPþ toxicity links with ATP depletion. The power of Caenorhabditis elegans as a model system lies with rapid and powerful genetic interaction studies to identify protein interactors capable of modifying a-syn action. An RNA interference screen to identify critical proteins involved in a-syn
protection identified a number of proteins involved in the endocytic pathway in addition to chaperones and other proteins. The usual caveat with a-syn–overexpression
model systems, but particularly for models involving organisms that do not natively express an a-syn–like protein, is that overexpressed a-syn might not adopt physiologically relevant cell functionality and that the cell impairment or deficiency has no overlap with the dysfunction occurring in PD. Thus, lower organisms seem an ideal tool for hypothesis generation but ultimately require translation to mammalian systems. Successful examples of translation from yeast to mouse models have highlighted new pathways with potential therapeutic targets.
Similar to nematodes, Drosophila does not possess clear homologues to a-syn. In models that involve overexpression of human a-syn, flies demonstrate loss of dopamine neurons associated with progressive loss of motor dysfunctions and
the presence of filamentous intraneuronal inclusions.
These flies also exhibited age-dependent retinal degeneration and premature loss of climbing activity. Induction of chaperone pathways rescues cells from the apparent effects of a-synuclein expression. The reproducibility of a-syn– induced dopaminergic cell death in flies has been a matter of contention among different laboratories, with some groups reporting cell shrinkage due to a-syn overexpression in particular dopamine neuron clusters that may be masked as cell loss when counted by using particular methods. The utility of Drosophila models of a-syn overexpression remains in question until the technical issues that prevent an understanding of phenotype are clearly defined.
As opposed to reports of dopaminergic cell death in the worm and fly, mouse transgenic models overexpressing human a-syn have not yet demonstrated overt degeneration in substantia nigra neurons. Transgenic mouse models driving
a-syn with various promoters such as PDGFb, mouse thymus cell antigen 1:Thy1, TH promoter, and prion (PrP) promoter have been described. These transgenic animals demonstrate markedly different phenotypes, making broad-based conclusions difficult
to draw. In mice overexpressing human a-syn and a-syn with PD-associated mutations driven by the PrP promoter, phenotype is related to dose, and the PD-mutation A53T
demonstrates greater in vivo neurotoxicity as compared with other variants; moreover, these mice develop adult-onset neurodegenerative disease with a progressive motoric dysfunction leading to death. Transgenic animals further
demonstrate an early phenotype before pathologic lesions form. Other important observations from transgenic mouse experiments include loss of straital dopaminergic terminals in case of PDGFb promoter-WT–a-synuclein expression, decreased rotarod performance and the presence of detergent-soluble and -insoluble a-syn species in Thy1-promoter-WT and A53T-a-syn expression. Without neurodegeneration in the substantia nigra, the challenge lies with picking a phenotype among the plethora of observations robust enough to screen potential therapies for efficacy and
yet possess reasonable homology to mechanisms thought to underlie pathogenesis in human PD. The lack of Lewy body formation in transgenic mice and selective degeneration of substantia nigra neurons might disqualify existing transgenic
mice as an appropriate model system for therapeutic testing. The next generation of transgenic might include conditional and regionally specific expression, or crosses of existing transgenics to mice that modify expression of a critical a-syn
modifier. As opposed to that of traditional transgenic mice, neurodegeneration in the substantia nigra due to a-syn expression via viral-vector–based delivery has been described in both rats and mice. Viral-based gene transduction in living animals results in acute and targeted gene expression, so-called somatic transgenics. Adenoassociated viral (AAV) vectors and HIV-1–derived lentiviral
vectors successfully direct high-levels of a-syn expression and loss of nigral and dopaminergic neurons in rodents. Co-delivery of the early-onset PD–associated protein
parkin prevents dopaminergic degeneration, but in the same model, delivery of GDNF does not prevent neurodegeneration. The authors speculate that GDNF treatment cannot modulate the cellular toxicity related to mutant a-syn accumulation. Virus-based models suggest a-syn as a viable target for therapeutic intervention. Whether a-syn viral transduction in rodents represents a viable in vivo model in which therapeutic
approaches prove efficacy remains speculative. Issues including a high technical proficiency requirement for model implementation, high variation between experiments, interlaboratory variation in reproducing the critical cell death
phenotype, and a high degree of labor in counting cells by stereology all prevent widespread use of the model system. Further, the lack of cell death in traditional transgenics might translate to a more cautious approach in interpreting viral transduction experiments, in which inflammation or viral transduction pathways may provide a necessary ‘‘second-hit’’ in causing cell death that may or may not have relevance to PD. Alternatively, acute somatic transgenics may not have
compensatory pathways that block cell death in the traditional transgenics. Transgenics that conditionally and acutely upregulate a-syn to the levels obtained through viral transduction will help resolve the issues and may provide the most
powerful model system.

Sorry for the dealy

2009-10-19T08:50:00.001-05:00

I was busy in between with some personal things and also on research front for a paper, which finally got published in JBC. Could not do the blog religiously for a while: but back again now.

In virto model systems for alpha-synuclein

2009-07-29T11:15:00.001-05:00

Cell-culture models of a-synuclein provide a valuable route for studying the physiologic and pathologic functions of the protein because they would be potentially amenable to high throughput translation to identify therapeutic compounds. a- Syn can be highly overexpressed in various cell lines and primary neuronal culture systems. Expression of a-syn containing a PD-associated point mutation in PC12 cells leads to loss of dopaminergic release, alteration of the ubiquitin dependent degradation system, and autophagic-dependent cell death. Overexpression of a-syn in H4-glioma cells results in an upregulation of markers associated with toxicity. Delivery of a-syn via HSV-1 transduction in mesencephalic
primary cultures likewise results in enhanced toxicity in infected cells. However, the goal of achieving a robust model of a-syn–dependent cell death in cultured cells amenable to translation to high-throughput screening remains an elusive goal.
Most described a-syn–overexpression systems in mammalian cells result in very mild to no significant changes in markers of toxicity associated with wild-type a-syn expression. Some culture systems demonstrate neuroprotective mechanisms due to a-syn expression. a-Syn expressed in NT-2D1 (human teratocarcinoma cell line) and SK-MC (human neuroblastoma cell line) cells delays cell death induced by serum withdrawal, but the effect is reversed via MPPþ (1-methyl-4-phenylpyridinium) exposure. In lieu of overt changes in cell-death markers associated with a-syn expression, numerous studies have described potential early phenotypes with possible relevance to pathogenesis. A kinetic basis for intracellular accumulation has been demonstrated by
overexpressing Flag- and His-tagged versions of a-syn in PC12 and SH-SY5Y cells. Demonstration of a-syn expression and oligomeric intermediates in living cells through bimolecular fluorescence complementation may also provide insight into a-syn mechanisms. Because overt cell death caused by a-syn expression has been difficult to achieve in mammalian cells, the future of in vitro model systems will likely involve phenotypes outside of toxicity but focused on particular aspects of a-syn conformation, association, or localization. The relevance of data developed in vitro will require confirmation in successful in vivo model systems.

Alpha synuclein Target Validation

2009-07-27T10:48:00.000-05:00

The majority of therapies for human disease involve the inhibition of a particular target important for pathogenesis. Many clinical trials fail because the target itself is not a critical component of disease. As a result, pharmaceutical companies
now demand more-stringent target-validation studies. Pathologic evidence from human PD tissue nominating a-syn as the primary agent of pathogenesis is circumstantial, whereas
human genetic studies firmly place the causative basis with asyn, but only in a very small proportion of familial and early onset- disease cases. The idea that a-syn is a robust therapeutic target in PD is provocative but nonetheless requires extensive
proof from target-validation studies. More than a decade of research centered on a-syn highlights outstanding questions through the descriptions of numerous model systems.

Alpha synuclein aggregation: mechanism and role in PD

2009-07-24T13:37:00.003-05:00

As a-syn is the most abundant protein composing the proteinaceous aggregates that define PD on a pathologic level, intense efforts revolve around understanding a-syn
accumulation and aggregation. a-Syn may assume oligomeric species through unknown mechanisms, and higher-order a-syn structures usually correlate with a-syn–dependent toxicity in cells (Fig). The specific conformational entities responsible
for protection, toxicity, and / or aggregation remain elusive. Factors known to modify a-syn aggregation and/or oligomerization include alterations of the primary amino acid sequence (e.g., PD-associated mutations); c-terminal truncations,
interactions with metal ions; interactions with Ab peptide; interactions with chaperone proteins such as Hsp70; interactions with apolipoprotein E, as well as
neurotoxins, pesticides, and herbicides; organic solvents; tyrosine nitration; phosphorylation; methionine oxidation; monoubiquitination; interaction with
polyanions and polycations, oxidative dimmers, and oligomers; histones; transglutaminase;and other protein–protein interactions.
As in other neurodegenerative disorders and diseases associated with protein aggregation, the role of organized inclusion bodies localized to disease-associated regions in PD affected tissue remains hotly debated. On the one hand, the
inclusion-body organelles may serve to sequester molecules that, for whatever reason, can no longer route through normal metabolism that would otherwise cause cellular dysfunction.
Experimental evidence supports this notion, in which the lack of a-syn–inclusion formation associates with toxicity. In contrast, the overt formation of inclusions may represent the toxic insult itself composed of molecules that were otherwise
nontoxic until association with the inclusion. In the first case, promoting inclusion bodies should facilitate neuroprotection. In the second case, inhibiting inclusion bodies would promote cell toxicity. Both situations may be true,
depending on the particular component of the inclusion body in question; for example, sequestration of toxic a-syn species into inclusion bodies may represent cytoprotection, with the cost of sequestering and inactivating other molecules necessary for normal function.
Therapeutically oriented efforts should focus on farther upstream events as opposed to modifying preexisting inclusion bodies, although this may be a moot point because model systems that demonstrate inclusions with morphologic similarity to Lewy bodies have yet to be developed. One major obstacle to understanding Lewy bodies is the failure to recapitulate the basic morphologic characteristics of Lewy bodies
in the context of relevant model systems. Although several model systems describe a-syn– and ubiquitin-positive aggregations in some fraction of cells, none can be
considered similar to the highly ordered structures found in PD tissue.
As a potential therapeutic target, the accumulation of a-syn into insoluble protein inclusions seems an important event in pathogenesis. A strong case can be made for therapeutically promoting inclusion formation in disease to protect cells from
more-soluble toxic species, as well as therapeutic approaches to dissolve inclusions to relieve the cells of a deleterious organelle that disrupts normal function. The dichotomy may not be resolved in the near future and hinders a-syn– antiaggregation strategies as a viable therapeutic approach.

Alpha synuclein: Primary Structure

2009-07-23T13:37:00.002-05:00

a-Syn is one of the most abundant ‘‘natively unfolded’’(intrinsically disordered) proteins that likely have different morphologies, including protofibrils, oligomers, and fibrils. Under physiologic conditions, a-syn has little or no ordered structure and possesses noteworthy conformational plasticity. The primary structure of a-syn is characterized as follows: (a) Residues 1-60 constitute the N-terminal domain
encompassing the conserved imperfect hexameric repeats (KTKEGV) and includes three missense mutations associated with early-onset familial PD (A30P, E46K, and A53T). The conserved 11-residue (XKTKEGVXXXA, X = any amino acid)repeat forms amphipathic secondary a-helical structures on binding to acidic phospholipid membranes, typical of the lipid-binding domains of apolipoproteins; (b) the central region (residues 61 to 95) is composed of an extremely hydrophobic, highly aggregation-prone NAC (nonamyloid component) sequence; and (c) the hydrophilic C-terminal region consists of highly acidic residues glutamic and aspartic acid, as well as proline, and is responsible for chaperone function.

Alpha Synuclein: Expression, physiologic function, and interacting molecules

2009-06-15T16:03:00.003-05:00

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 Cu2þ 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.

Focus on another PD protein: Alpha -Synuclein (a-syn)

2009-06-12T08:53:00.000-05:00

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.

LRRK2 Kinase activity: Tergetting the kinase

2009-06-05T11:48:00.002-05:00

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.

In vivo models of LRRK2

2009-06-03T09:16:00.002-05:00

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.

in-vitro models of LRRK2

2009-05-14T10:30:00.001-05:00

LRRK2 protein is relatively rare in cells compared with other kinases, and overexpression of the human wild-type protein in cell lines or primary neuronal cultures is well tolerated, with relatively minimal increases in markers of cell
death or other morphologic indicators. In contrast, overexpression of wild-type mixed-lineage kinase 2 protein, the closest kinase to LRRK2 apart from LRRK1, based
on sequence similarity of the kinase domain, results in immediate apoptosis. However, overexpression of LRRK2 protein with the G2019S mutation results in a significant increase in markers of cell death, including neurite shortening
and changes in membrane permeability. The effects appear to track with kinase activity, because mutations that ablate kinase activity also nullify the upregulation
of markers of toxicity associated with LRRK2-G2019S expression.A phenotype derived from WT-LRRK2 protein expression is more subtle, but expression of WT-LRRK2 protein in neurons may leave cells more susceptible to other toxicities, including peroxide exposure. Perhaps a more relevant phenotype for target validation would derive from the knockdown of LRRK2 protein or ablation of LRRK2 kinase activity in concert with exposure to agents commonly used in models of PD, such as overexpression of human a-syn or chemical toxins selective for dopaminergic neurons. However, numerous technical challenges include the lack of commercially available antibodies
with sufficient specificity to detect human and (more so) mouse LRRK2 protein, coupled with incomplete knowledge of the half-life of the endogenous protein in primary cells that constitute in vitro models. The criteria for relevant in vitro models of PD are still hotly contested because the defining end-point requirements such as cell death, protein aggregation, or other biochemical changes in signaling or metabolism are not agreed on. In vitro data that demonstrate kinase-dependent changes in LRRK2 overexpression paradigms are provocative but not necessarily
relevant, unless the endogenous and physiologically important targets for kinase activity are verified in these systems. The fact that a phenotype is observed for PD-associated mutations that link with kinase overactivity does not imply
that the protein is correctly functioning in these cells. Ultimately, in vivo model systems that demonstrate LRRK2-dependent phenotype more universally agreed as important for PD (neurodegeneration and inclusion formation in dopaminergic
neurons within the SNpc in intact animals) may be required before in vitro model systems can be validated as useful tools for validating therapeutic compounds before use in humans.

LRRK2 target validation

2009-05-07T15:44:00.001-05:00

The implication of LRRK2 as a potential target for neuroprotection strategies in PD clearly originated from human genetic studies. The LRRK2 protein had not been previously identified as a critical mediator of cell-death pathways or mechanisms important in neuron survival before the identification of mutations causative of PD. Further, most individuals with PD do not harbor known LRRK2 mutations, and
missense mutations are relatively rare in accounting for a small percentage of PD in most populations. Model systems that demonstrate an LRRK2-dependent phenotype are necessary to validate LRRK2 as an appropriate potential therapeutic target and to help address whether LRRK2 would serve as a general target for therapy in PD, because most individuals living with PD presumably do not harbor coding mutations in LRRK2. Thus, the question of whether LRRK2 represents a valid target in PD cases without LRRK2 mutations may not be fully answered until safe and effective modifiers of LRRK2 action are available in the clinic.

Enzymatic activity of LRRK2

2009-04-28T16:31:00.002-05:00

LRRK2 harbors both a GTPase and kinase domain, an extremely rare arrangement found only in LRRK1 and possibly the DAPK1 protein in mammals. A solid precedent for
GTPase activity dependent modification of protein kinase activity suggests that the enzymatic domains encoded inLRRK2 may function as a self-regulatory apparatus. The most common LRRK2 mutations localize to the kinase domain (amino acid G2019) and the GTPase domain (amino acid R1441), implicating enzymatic output as critical to PD. Beyond a domain-prediction analysis that helps define conserved features and potentially critical residues, in silico approaches do not further predict LRRK2 function in cells. The LRRK2 kinase domain possesses highest sequence homology
to the mixed-lineage kinase (MLK) subfamily of MAPKKK proteins but differs in the critical amino acids that define MLK proteins, whereas the GTPase domain displays
distinct architecture reminiscent of Rab-like GTPases. As one of the largest protein kinases in the mammalian kinome, LRRK2 will necessarily resist characterization by the usual gauntlet of biochemical assays that have historically well served the characterization of other protein kinases. Nevertheless, tremendous advances in a short time have outlined a surprising consensus story for the impact of PD causing
mutations on protein function. Before a functional description of the LRRK2 protein, a functional description of LRRK1 suggested that intrinsic kinase activity was stimulated on GTPase activation. This phenomenon holds true for LRRK2 as well, in which application of GTP or GTPgS spurs modest increases in kinase output. However, a clear relation between kinase activity and GTPase activity is defined through the us of specific mutations that ablate either GTPase activity, kinase activity, or both activities. Mutations in the conserved ATP binding pocket site, in the consensus residues of the activation loop, or mutation within the proton-acceptor site have no apparent effect on GTP binding. In contrast, mutations in the
GTPase domain thoroughly ablate kinase activity. Thus, LRRK2 functions as a signal-transduction pathway encoded into a single protein. The activity of LRRK2 is
perhaps an ancient design, because the orthologue GbpC in slime mold operates in a similar manner, although GbpC also encodes GEF domains upstream of GTPase activity.
Through functional descriptions of GbpC, LRRK1, and LRRK2 protein, the encoded domains function in a signal F2c cascade ultimately to regulate kinase output.In an initial functional description of the LRRK2 protein, the most common PD-associated mutations, G2019S and R1441C, enhanced kinase activity but failed to alter other
basic biochemical properties, such as protein localization and turnover. PD-causing mutations in the GTPase domain likewise enhance the proportion of GTP-bound protein in pull-down experiments, whereas mutations in the kinase domain have no effect on the proportion of GTP-bound protein. Additional studies demonstrate that PD-causing
mutations in the GTPase domain do not result in a higher affinity for GTP; rather, GTP hydrolysis is disrupted because of PD-associated mutations, thereby prolonging GTP-bound states and an activated GTPase domain. Missense mutations in the LRRK2 gene that segregate with disease in families and are therefore likely pathogenic variants all result in enhanced kinase activity in vitro, although not all
laboratories demonstrate a significant difference between kinase activity associated with wild-type protein and that containing PD-causing mutations. Differences in assay
protocols and the lack of a relevant substrate and robust kinase-dependent phenotype in cells combine to prevent the assumption that kinase output is the true and only possible functional link between LRRK2 protein and PD. However, the available data, when taken as a whole, suggest that kinase activity represents the final output of activity responsible for pathogenesis.

LRRk2 expression and localization

2009-04-27T17:03:00.001-05:00

LRRK2 mRNA displays near ubiquitous localization throughout the mouse and human brain, with particular concentration within neurons of the cortex, striatum, and
hippocampus. Protein distributes in a likewise manner, detectable in the vulnerable neurons of the substantia nigra pars compacta. LRRK2 displays particularly high expression in the kidney and appears to increase in expression on organogenesis and cell maturation. LRRK2 adopts a punctate intracellular cytosolic localization
that associates with various membranous structures including vesicles, mitochondria, Golgi, and the ER. Biochemical fractionation suggests that LRRK2 resides on the
cytoplasmic side of membrane-containing organelles without evidence of nuclear or mitochondrial internalization. Similar to a-syn, clear orthologues to LRRK2 have been
identified in all described mammalian genomes, but in invertebrates, orthologous genes may show closer homology to mammalian LRRK1 than to the LRRK2 gene. Close sequence homology and overlapping expression profiles between the LRRK1 and LRRK2 genes suggests redundancy in function. LRRK2-deficient mice are viable with no dramatic abnormalities, although detailed reports have not been published. Loss of the LRRK orthologues in Drosophila seems to produce a strain-dependent phenotype, and additional studies will help resolve the controversy. Loss of
LRRK orthologues in nematodes produces defects in vesicle sorting, whereas loss in slime mold produces defects in chemotaxis.

LRRK2 structure and function

2009-04-23T17:11:00.000-05:00

As opposed to a-syn, LRRK2 had not been cloned or thoroughly annotated when missense mutations were identified in PD cases. Bioinformatic prediction strongly suggested that LRRK2 encodes a GTPase-like domain together with a kinase domain, thereby placing LRRK2 within the fraction of the proteome considered modifiable with small molecules or intervention therapies. The potential for LRRK2 as a therapeutic target depends not only on activities of the protein associated with PD, but on normal function and the relation between health and disease. For example, generalized inhibition of LRRK2 activity may not be compatible with normal cellular function, or inhibition of one particular aspect of LRRK2 activity may not influence another activity that underlies
disease mechanisms. Deep insight into LRRK2-related biology will provide the background necessary for rationally designed therapeutic approaches.

Autosomal dominant genes in PD

2009-04-22T13:29:00.002-05:00

LRRK2.
A genetic locus initially described as associated with late-onset PD in a large Japanese kindred demonstrated association with late-onset autosomal-dominant PD in several additional families, and missense mutations in the LRRK2 gene were identified. Sequence analysis of the encoded enzymatic domains revealed an alteration within the activation loop of the kinase domain responsible for a
significant percentage of PD disease in many case populations studied. In general, common genetic variation in LRRK2 does not appear to contribute heavily to PD susceptibility, with the exception of some Asian populations, but in some populations, the missense mutations themselves account for more than one third of PD cases. Mutations that clearly segregate with disease in well described
families and are overrepresented in PD cases versus age-matched controls represent a fraction of described LRRK2 variants, and the difficulty in distinguishing benign versus pathogenic variants prevents resolution of the true frequency
of LRRK2 mutations in PD.

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 PD does 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.

Autosomal-recessive parkinsonism

2009-04-21T15:22:00.001-05:00

Families that inherit PD in a manner compatible with autosomal-recessive disease have long been described, with disease usually manifesting earlier than that in the majority of PD cases. Mutations in the parkin gene were described in
Japanese juvenile-onset cases of disease, and genetic variation in parkin clearly associates with early-onset PD in multiple ethnicities. Apart from the large deletions and rearrangements that inactivate expression, pathogenic point
mutations impart deleterious function compatible with a general loss-of-function disease mechanism. Likewise, loss-of-function mutations that include nonsense
mutations, genomic rearrangements, and missense mutations AU1c have also been described in the PINK1 (PTEN-induced kinase-1) and DJ-1 genes in early-onset cases. In general, overexpression of these proteins usually imparts cytoprotective
properties to cells from a variety of insults. Because most effective drug therapies for a variety of human illnesses involve the ablation or reduction of activity of associated targets, dealing with loss-of-function mechanisms usually imparts additional technical demands on therapeutic strategies. Although the autosomal-recessive genes encode clearly relevant proteins in PD, additional work that characterizes underlying cellular pathways in relevant disease models seems necessary before parkin, PINK1, and DJ-1 can be fully realized as potential therapeutic targets.

Genetic Susceptibilities in Parkinson’s Disease

2009-04-20T13:46:00.000-05:00

The current understanding of PD includes a long initial
phase, often dubbed ‘‘preclinical,’’ in which non–movement disorder
symptoms are common but heterogeneous in presentation. Early twin studies suggest that genetic susceptibilities might not play a large role in PD etiology. More
recent studies show that, in some populations, a single point
mutation in the LRRK2 gene causes more than one third of PD
cases, with the ancient G2019S mutation reported at a higher
frequency in patients without knowledge of PD in their family
than in patients aware of a family history of disease, in some
populations. Genetic studies have therefore illuminated
specific causes of PD that promise to aid in the delineation
of pathogenesis and the development of therapeutics.

3 prominent questions on LRRK2

2009-04-03T11:07:00.002-05:00

A recent review article by Cookson et al have pointed out 3 prominent questions about LRRK2. These are some burning issues and answers to these questions would lay the fundamental foundation stone in understanding the LRRK2 biology at the molecular level in a better way. It will also help in designing therapeutics for LRRK2, which itself has been a good kinase target.
The 3 questions that the authors fundamentally wish to address are:

1. What is the normal function of LRRK2.

2. How mutations impact LRRk2 function.

3. Possible mechanisms of LRRK2 mediated neurotoxicity.

Glutamate Receptors and PD ?

2009-03-26T19:14:00.002-05:00

Metabotropic Glutamate Receptors (mGluRs) have been shown to be involved in PD and are projected as important therapeutic targets with a neuroprotective approach for this aging disorder. The major focus for this class of GPCRs in its role in PD is through the non-cannonical non-dopaminergic pathway. Loss of Dopaminergic neurons in PD results in significant changes in other neurotransmitter systems (Glutamate and GABA) that play key roles in mediating mediating normal basal ganglia functions.

In a MPTP model of monkey, it has been demonstrated that antagonist MTEP (antagonist to mGluR5) shows anti-parkisonian effect and offers some degree of neuroprotection. This antagonist molecule is known to cross the blood-brain barrier and offer neuroprotection and this drug is cleared or flushed out very quickly. However, it is not clear what is the molecular mechanism of this mode of action. And the other question that crosses my mind is that how does this MTEP molecule really functions ? does it couple to the GPCR and induce the heterotrimeric G-protein signaling ? These are open questions and need to be explored more to figure out the mechanistic pathway.

GPCR crystallization : trick or science

2009-03-23T08:55:00.002-05:00

The scientific community has been wondering about the know how for GPCR crystallization. It has took decades to arrive at the first structure of a GPCR: bovine rhodopsin. Though the structure was deciphered in 2000, the structure represented only an inactive form of the protein. Time took its toll and it was after another 8 years of hard work and dedication by brilliant scientists that the second structure of GPCR (the beta-2- adrenergic receptor) was published in 2008. The trick involved in introducing T4-lysozyme fused within the i3 loop of the receptor molecule to aid the stabilization of detergent solubilized receptor as well as the crystallization phenomenon. This structure also had the high affinity antagonist carazolol bound to it and thus defined the overall architecture of beta2AR and the structure of the ligand-binding pocket.

The Therapeutic Potential of LRRK2 and Alpha-synuclein in Parkinson's Disease

2009-03-18T16:06:00.002-05:00

My review article with Dr. West just hit the Pubmed search today.....the full PDF is yet to be there....The article gives a broad overview on LRRK2 and alpha-synuclein with their potential role in Parkinson's disease and the therapeutic potential of these. The abstract of our article is as follows: Current treatments for Parkinson's disease fail to modify disease progression and the underlying pathogenic mechanisms remain elusive. The identification of specific targets responsible for disease will aid in the development of relevant model systems and the discovery of neuroprotective and neurorestorative therapies. Two promising protein candidates, alpha-synuclein and LRRK2, offer unique insight into the molecular basis of disease and the potential to intervene in pathogenesis. Although multiple lines of evidence support alpha-synuclein and LRRK2 as robust targets for therapy, the connection between protein function and neurodegeneration is unclear. Technology capable of mitigating alpha-synuclein and LRRK2 disease-associated function will ultimately be required before the true value of these proteins as therapeutic targets can be discerned.

GPCR activation and FRET

2009-03-16T10:28:00.002-05:00

A recent mini-review article on G-protein and GPCR activation involving FRET study was nicely described by Vilardaga et al (Mol Endocrinol. 2009 Feb). It is a nice paper giving background on GPCR as well as the principles of FRET and describe the mechanisms of GPCr activation using this technology. The abstract of the paper states that : Many biochemical pathways are driven by G protein-coupled receptors (GPCRs), cell surface proteins that convert the binding of extracellular chemical, sensory, and mechanical stimuli into cellular signals. Their interaction with various ligands triggers receptor activation that typically couples to and activates heterotrimeric G proteins, which in turn control the propagation of secondary messenger molecules (e.g. cAMP) involved in critically important physiological processes (e.g. heart beat). Successful transfer of information from ligand binding events to intracellular signaling cascades involves a dynamic interplay between ligands, receptors, and G proteins. The development of FRET and BRET-based methods has now permitted the kinetic analysis of initial steps involved in GPCR-mediated signaling in live cells, and in systems as diverse as neurotransmitter and hormone signaling. The direct measurement of ligand efficacy at the level of the receptor by FRET is also now possible, and allows intrinsic efficacies of clinical drugs to be linked with the effect of receptor polymorphisms.

Finally Genentech and Roche shake hands

2009-03-13T10:58:00.002-05:00

Directly quoted from news briefing as appeared at Yahoo Business:

Roche has agreed to acquire the 44 percent of shares in the U.S. firm it does not already own for $95 each, ending a long pursuit of the U.S. biotech group and its lucrative cancer drugs.

Even if clinical trial results with Genentech's cancer drug Avastin disappoint next month, the purchase of the world-leading U.S. biotech group makes sound financial sense.

Genentech is at the cutting edge of both biotechnology and cancer medicine -- exactly the place where all big drugmakers want to be as the flow of traditional drugs from research labs stalls and patents of today's blockbusters expire.

Big drugmakers have been seeking to diversify and reduce their reliance on slow-growing traditional prescription medicines, which face patent expiries and falling prices.

Roche's play for the remainder of Genentech will also yield synergies -- some $750-850 million a year before tax, according to Roche -- but the big prize is maximum exposure to the fastest-growing section of the global pharmaceuticals market.

Why GPCRs and membrane proteins migrate anomalously on SDS-PAGE ?

2009-03-11T14:05:00.003-05:00

There has been a recent discussion about the migration pattern of membrane proteins and GPCRs in SDS-PAGE gels. From my experience on working with GPCRs for greater than 7 years, these proteins may a times have a tendency to run anomalously in SDS-PAGE gels.

Recent report explains the anomalous behavior being attributed to detergent. The abstract of the paper describes as: Migration on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) that does not correlate with formula molecular weights, termed "gel shifting," appears to be common for membrane proteins but has yet to be conclusively explained. In the present work, we investigate the anomalous gel mobility of helical membrane proteins using a library of wild-type and mutant helix-loop-helix ("hairpin") sequences derived from transmembrane segments 3 and 4 of the human cystic fibrosis transmembrane conductance regulator (CFTR), including disease-phenotypic residue substitutions. We find that these hairpins migrate at rates of -10% to +30% vs. their actual formula weights on SDS-PAGE and load detergent at ratios ranging from 3.4-10 g SDS/g protein. We additionally demonstrate that mutant gel shifts strongly correlate with changes in hairpin SDS loading capacity (R(2) = 0.8), and with hairpin helicity (R(2) = 0.9), indicating that gel shift behavior originates in altered detergent binding. In some cases, this differential solvation by SDS may result from replacing protein-detergent contacts with protein-protein contacts, implying that detergent binding and folding are intimately linked. The CF-phenotypic V232D mutant included in our library may thus disrupt CFTR function via altered protein-lipid interactions. The observed interdependence between hairpin migration, SDS aggregation number, and conformation additionally suggests that detergent binding may provide a rapid and economical screen for identifying membrane proteins with robust tertiary and/or quaternary structures.

WIll the deal of Roche & Genentech see the light of the day ?

2009-03-10T14:12:00.002-05:00

Following the encouragement of the buyout of Scheing-Plough by Merck, the drug giant Roche once again stirred up the discussion of acquiring Genentch. The two companies had earlier conversations which fell through due to a solid consensus on the pricing issue. Roche has now offered a price tag of $93 per share. If they still bump up the value by two dollar per share, the deal would be valued at $46.7 billion. Genentech authorities are looking for a value of more than $100 per share.

We will wait and watch how the deals goes and if at all it goes or not. The likelihood of it making through is rather high is what my feeling is.

Merck and Schering-Plough consolidate operations through acquisition

2009-03-09T19:18:00.002-05:00

Drug maker giant Merck shedding off prior controversial ventures of Vioxx has announced the acquisition of rival drug maker Schering-Plough for a whooping $41 billion. The new deal will help consolidate the drug industry more on a solid footing. Perhaps, this news is a blow for the sales and marketing force as that sector might experience some lay-off resulting from this consolidation effort.

The new force will be in terms more effective in bringing discovery to a quicker platform with a renewed work force and bigger talent pool of brilliant scientists. The healthcare industry will probably benefit more out of this with more drugs coming out from a single big pharmaceutical giant.

This new deal follows on the heals of the Pfizer - Wyeth and probably opens the door for the Roche - Genentech to fall into place.

A new Orphan GPCR

2009-03-06T13:56:00.002-06:00

The orphan g protein-coupled receptor 3 (GPR3) modulates amyloid-Beta Peptide generation in neurons.

Report convincingly showing the link between GPCR modulation and AD.

No ligand has yet been identified for this GPCR.

The abstract of the paper states: Deposition of the amyloid-beta peptide is a pathological hallmark of Alzheimer's disease. A high-throughput functional genomics screen identified G protein-coupled receptor 3 (GPR3), a constitutively active orphan G protein-coupled receptor, as a modulator of amyloid-beta production. Overexpression of GPR3 stimulated amyloid-beta production, whereas genetic ablation of GPR3 prevented accumulation of the amyloid-beta peptide in vitro and in an Alzheimer's disease mouse model. GPR3 expression led to increased formation and cell-surface localization of the mature gamma-secretase complex in the absence of an effect on Notch processing. GPR3 is highly expressed in areas of the normal human brain implicated in Alzheimer's disease and is elevated in the sporadic Alzheimer's disease brain. Thus, GPR3 represents a potential therapeutic target for the treatment of Alzheimer's disease.

Eukaryotic Membrane Proteins

2009-03-05T15:07:00.003-06:00

Membrane proteins are extremely difficult to overexpress in an heterologous system. While E.Coli serves as a perfect host for expression, various difficulties render this system as an inconvenient tool of use.

Bacterial expression based on Escherichia coli has been one of the most widely studied because of its ease of availability, handling and scale up; and furthermore it is cheap to grow. However, this system has turned out not to be suitable for expression of mammalian transmembrane proteins due to the lack of post-translational modifications (e.g. glycosylation, fatty acid acylation, and phosphorylation) and the processing events required for correct folding of mammalian proteins. The
accumulation of expressed proteins in inclusion bodies (in a misfolded state) is yet
another drawback, as it is very difficult to recover functional protein. In addition,
insertion of the recombinantly expressed mammalian proteins into bacterial membranes
has caused toxicity in host cells.

YEASTS
Yeasts are unicellular eukaryotes which can easily be grown in large-scale cultures. They combine the advantages of short generation times (2 h) and growth in simple media. Easy handling and rapid genetic manipulation and the potential to perform eukaryotic post-translational modification renders yeast as one of the organisms of choice for over-expression. High levels of heterologous expression in yeast have been obtained with β2-adrenergic receptor, with the expression level being 115 pmol/mg of the membrane protein, for an O.F. 23. However, the level of expression of other GPCRs expressed in this system varies between 0.02 pmol/mg and 40 pmol/mg. Though yeast has been rather widely used for production of membrane proteins and GPCRs, there are four important issues. First, the yeast is surrounded by a tough cell wall which must be disrupted in order to extract the membranes and
intracellular proteins. In addition, protease-deficient strains should be used and protease inhibitors should be included during breakage of the cells. Second, the lipid composition of yeast membranes is different from that of mammalian cells. The low cholesterol content or even the presence of ergosterol results in altered binding properties of the expressed membrane proteins. Third, though the yeast system is capable of performing eukaryotic post-translational modification, the composition and quantity of N-glycans added by yeast are different from that of mammalian cells, and this can be problematic when specific oligosaccharide structures are essential for functionality. (Fourth and) finally, during large scale production, an appropriate ligand should be added to the growth medium and the pH should be maintained at the optimal value for the receptor in study.

A few other systems have been used for the expression of membrane proteins and GPCRs. Archaebacteria such as Halobacterium salinarium have been used to express two receptors successfully, the β2-adrenergic receptor and the yeast α-mating factor receptor. H.salinarium has the capacity to express large quantities of bacteriorhodopsin; and it was assumed that this system could express other transmembrane mammalian proteins. However, this was not the case and even chimeras of bacteriorhodopsin and mammalian transmembrane proteins resulted only in modest expression levels.

In addition to Baculovirus/insect cell, there are other insect systems.
For instance, the extracellular part of the luteinizing hormone (LH receptor) has been expressed in a non-functional form in a caterpillar expression system. In addition, there is a new system for expression of GPCRs in fly eyes. The Drosophila melonogaster metabotropic glutamate receptor (DmGluRA) was expressed by this novel strategy in good yields (170 μg/g fly heads) and was purified to homogeneity.

Insect Cells / BACULOVIRUS

The insect cell based baculoviral system provides an alternative approach for expression of GPCRs. The advantage of the baculovirus expression system is that it expresses the majority of membrane proteins and GPCRs in a functional form and at levels 10-100 fold higher than those observed in cells and tissues endogenously expressing them. In this system, the protein production results from infection of insect cells by recombinant viruses encoding the gene(s) of interest. The Baculovirus Autographica californica multiple nuclear polyhedrosis virus (AcMPPV) has been most widely used for the expression of membrane proteins in insect cells (usually using Spodoptera frugiperda, Sf9 cells). In most cases, the expression is driven by the polyhedrin or the p10 promoter, both of which are ‘very late’ promoters and are switched on only about 24 h post viral infection. The expression of the recombinant proteins under the control of very late promoters generally peaks 48-72 h post infection. The virus undergoes a lytic cycle and the cells die about 4-5 days post infection. The insect cells are easy to grow, can adapt to serum-free media and growth can be scaled up with ease. These cells can also perform post-translational modifications such as fatty acid acylation, phosphorylation and glycosylation. The recombinant proteins exhibit characteristics very similar to their native counterparts. In addition, co-expression of GPCRs and mammalian G-proteins is possible in this system allowing in vivo analysis of the specificity of interactions with G protein subtypes. A large number of membrane proteins and GPCRs have been successfully expressed using this system with levels as high as 10-
100 pmol/mg of membrane protein. The receptors which were recombinantly expressed
at such high levels include β-adrenergic, serotonin, muscarinic, dopamine, neurokinin
and chemokine receptors.
The expression levels sometimes depend on receptor subtype as well as the presence and position of a fusion tag. One drawback of this system is that it
often gives rise to heterogeneous proteins, arising out of poor glycosylation, which results in altered binding properties. Also, due to low cholesterol abundance in insect cells, alteration in GPCR binding has been observed with oxytocin receptor. Coexpression of various chaperones, peptidases, foldases and glycosylating enzymes has been shown to improve the secretion, processing and glycosylation of heterologous
proteins expressed in the baculovirus system.

GAPS and GEFs for LRRK2

2009-03-05T11:04:00.002-06:00

As it is known that LRRK2 kinase activity is controlled by GTPase activity, there is a general query about the role of GEFs and GAPs in turning on/off of the GTPase activity. The partial crystal structure of LRRK2 - ROC domain indicates that the protein forms a dimer through generated by extensive domain-swapping. PD-associated mutants (R1441 and I1371) located at the interface of the two monomers provide exquisite interactions to stabilize the ROC dimer.

However, with the minimal quantity of LRRK2 expressed, it is not known which GEF's or GAPs play a role in the GDP/GTP exchange. There has not yet been and GDP-locked or GTP-locked structure / mutant of LRRK2 known. If someone comes up with either or both of them, a yeast-two hybrid approach can be used to find out the GAPs or GEFs.

LRRK2 story back again from my side

2009-03-05T11:01:00.003-06:00

Keystone Symposium: Neurodegenerative Diseases: New Molecular Mechanisms

I was at this symposium very recently and got to hear very nice talks. The focus on the talk was mostly AD and HD and less emphasis on PD. The quality of presentations and the posters were very nice and of high scientific depth.

Several pharmaceutical companies were interested in LRRK2 poster of mine: looks like LRRK2 has been out there as a very attractive therapeutic drug target. And being a kinase, it attracts people very much to invest and investigate in order to arrive at a putative inhibitor.

LRRK2 Quaternary Structure Influences Enzymatic Activity

2008-11-13T12:08:00.000-06:00

My poster at SFN:

Poster Session 342, Parkinson's Disease: Cells and Mechanisms II, which will be held on Monday, November 17, 2008 in the Washington Convention Center: Hall A-C. The session will begin at 8:00am

Abstract Title: LRRK2 quaternary structure influences enzymatic activity
Presentation Number: 342.6
Presentation Time: 9:00am - 10:00am
Poster Board Number: Q1

Abstract

The leucine-rich repeat kinase 2 (LRRK2) protein, as part of a ‘druggable’ class of proteins, represents a viable target for therapeutic modulation in Parkinson’s disease through the identification of small molecule kinase inhibitors. Mutations in the LRRK2 gene cause late-onset Parkinson’s disease clinically and neurochemically indistinguishable from typical idiopathic disease. In cells, we find that LRRK2 protein forms complex higher order conformations. Herein, we investigate the relationship between the oligomeric forms of the protein in the disease-associated mutants as compared to the wild-type protein. We genetically and pharmacologically dissect the effect of higher-order LRRK2 structures on kinase activity and neurotoxicity. Our results show that quaternary structure confirmations critically influence LRRK2 activity in cells.

Phosphorylation of LRRK2: Functional characterization

2008-11-10T14:46:00.002-06:00

The poster in SFN on the above topic by G. Ito et al talks about the N-terminal Serine residues (Ser 910 and Ser 973). The authors claimed in the abstract that mutating these residues did not alter the Kinase of GTPase activity of the protein. What it appears to me that these residues might not be the ones which governs the Kinase or GTPase activity in PD-related patients. I will look forward for more results in the meeting.

LRRK2 in SFN

2008-10-29T12:36:00.003-05:00

SFN (The Society for Neuroscience) Annual meeting is coming up in mid-November, at Washington DC, USA.
A lot of abstracts have been submitted for both poster and talk sessions. There are a couple of abstracts dealing with various aspects of LRRK2. I will subsequently discuss these abstracts and my views on them from now on: leading upto the meeting: which should eventually generate a healthy discussion when we meet all the presenters at the meeting.

LRRK2 Therapeutics

2008-10-06T16:00:00.002-05:00

The other significant gene which acts as a major player in the autosomal dominant PD is the LRRK2. It was first identified in a Japanese population in 2002 and first cloned in 2004.
The great significance of this gene is that it has both kinase activity and GTPase activity encoded within the same molecule. Literature report also suggests that the Kinase activity is governed by the GTPase activity, however, the GTPase activity is normal even if the kinase activity is ablated.
Targeting for small molecule inhibitors for ablation of kinase activity will be a possible therapeutic target for the disease.

Therapeutic targets for PD (Contd..)

2008-10-01T14:27:00.003-05:00

As discussed before, targeting alpha-synuclein for prevention of neurodegeneration and vying for neurorestoration would be a good therapeutic target. alpha-synulein aggregation has been one of the most widely cited cause leading to Lew Bodies and finally to Dopamineric cell death. Now: what causes the alpha-synuclein molecule to aggregate ? What is the mechanism of the aggregation ?
Being the most abundant protein comprising the proteinacious aggregates that define PD on pathologic level, intense efforts revolve around understanding alpha-synuclein accumulation and aggregation. alpha-synuclein may assume oligomeric species through unknown mechanisms, and higher-order alpha-synuclein structures usually correlate with toxicity in cells. What specific conformational entities are directly responsible for protection, toxicity and/or aggregation remain elusive. Factors known to modify alpha-synuclein aggregation and/or oligomerization include mutations in the primary amino acid sequence (e.g., PD-associated mutations: A30P, E46K, A53T), C-terminal truncations, interactions with metal ions, interactions with Aβ peptide, interactions with chaperone proteins such as Hsp40, Hsp70, interactions with apolipoprotein E, as well as neurotoxins, pesticides and herbicides, organic solvents, tyrosine nitration, phosphorylation, methionine oxidation, monoubiquitination, interaction with polyanions & polycations, oxidative dimers, histones, transglutaminase, and other protein-protein interactions, as well as other processes including mitochondrial dysfunction, oxidative stress, neuroinflammation, l-DOPA treatment and DA metabolism etc.

As a potential therapeutic target of action, the accumulation of alpha-synuclein into insoluble protein inclusions seems to be an important event in pathogenesis, yet a strong case can be made for therapeutically promoting inclusion formation in disease to protect cells from more soluble toxic species, as well as therapeutically dissolving inclusions to relieve the cells of a deleterious organelle that disrupts normal function. This dichotomy hinders alpha-synuclein as a viable target for therapeutics.

Therapeutic targets for PD

2008-09-30T15:42:00.002-05:00

As LRRK2 and alpha-synuclien are the two most important genes involved in the pathological manifestation of PD, these are the obvious two candidates which can be targeted for therapeutic purposes. The main approach would be to stop or slow the progression of the disease: and the best therapy possible would be complete reversal.

What are the aliases for LRRK2

2008-09-26T12:32:00.003-05:00

LRRK2 is known by a host of other different names: both the gene of the gene product. The commonly used aliases for LRRK2 are:

* DKFZp434H2111 * Dardarin
* FLJ45829 * LRRK2_HUMAN
* PARK8 * RIPK7
* ROCO2

Where is LRRk2 located ?

2008-09-26T12:31:00.002-05:00

If you have ever wondered where LRRK2 is found in the human chromosome:

Cytogenetic Location: 12q12

Molecular Location on chromosome 12: base pairs 38,905,085 to 39,051,869

In details:

The LRRK2 gene is located on the long (q) arm of chromosome 12 at position 12.

More precisely, the LRRK2 gene is located from base pair 38,905,085 to base pair 39,051,869 on chromosome 12.

What are your chances of inheriting PD

2008-09-25T10:29:00.002-05:00

If any member of your family has a history of PD, it is highly probable that you might be predisposed to the disease. The gene which has created a buzz in the scientific community for its role in PD is LRRK2. It is a relatively new gene and lots of scientific research is being carried out now and information is pouring in at a constant pace.
The mutation which has been prominent is the G2019S mutation on the LRRK2 gene found in chromosome 12. The single-letter change in the DNA code dramatically increases a person’s odds of developing Parkinson’s from one or two in a hundred to as much as eight in 10. One recent study found that a person who inherits the mutation has a 28% chance of developing Parkinson’s by the age of 59, 51% by the age of 69 and 74% by the age of 79 (Healy et al. 2008, Lancet, 7, 583-590).

Sergey Brin affected by LRRK2

2008-09-25T10:07:00.002-05:00

Google co-founder Sergey Brin has indicated that he is predisposed to PD through a family history and most probably the gene LKKR2 is involved in the same, with the mutation at G2019S.

http://news.cnet.com/8301-1023_3-10045958-93.html

LRRK2 in news

2008-09-25T09:54:00.002-05:00

Recently the PD field and specifically the LRRK2 gene came into limelight. The co-founder of Google, Sergy Brin has been diagnosed with PD and preliminary results point out to the fact that there is a point mutation in G2019, where the Glycine residue has been mutated to Serine (S) [G2019S mutation]. This mutation has been linked with enhanced levels of kinase activity and is known to be effected in PD: both in familial (5-6%) and sporadic cases (1-2%).

Brin’s mother was diagnosed with Parkinson’s, and now he has to live with the reality that he might one day develop the disease as well. Says Brin:

Nonetheless it is clear that I have a markedly higher chance of developing Parkinson’s in my lifetime than the average person. In fact, it is somewhere between 20% to 80% depending on the study and how you measure. At the same time, research into LRRK2 looks intriguing (both for LRRK2 carriers and potentially for others).

This leaves me in a rather unique position. I know early in my life something I am substantially predisposed to. I now have the opportunity to adjust my life to reduce those odds (e.g. there is evidence that exercise may be protective against Parkinson’s). I also have the opportunity to perform and support research into this disease long before it may affect me. And, regardless of my own health it can help my family members as well as others.

I feel fortunate to be in this position. Until the fountain of youth is discovered, all of us will have some conditions in our old age only we don’t know what they will be. I have a better guess than almost anyone else for what ills may be mine — and I have decades to prepare for it.

Also more info available at:

http://thegenesherpa.blogspot.com/2008/09/lrrk2-and-parkinson-disease.html

http://news.cnet.com/8301-1023_3-10045958-93.html

LRRK2

2008-09-22T14:41:00.004-05:00

LRRK2 (Leucine Rich Repeat kinase 2) is a cytoplasmic protein of 2527 amino acids. This protein is widely expressed in Substantia Nigra (SN) specially in the A9 Dopaminergic neurons (A9 DA). Other regions in the brain where LRRK2 has also been identified include cortex, hippocampus, straitum and cerebellum. Peripheral organs where the existence of LRRK2 has been detected include Heart, Placenta, Kidney, Lung, Skeletal Muscle, Pancreas etc. Subcellular localization of LRRK2 is observed in Golgi, Mitochondira and endosomal vesicles.

As a protein, LRRK2 has both GTPase activity and kinase activity encoded by the same molecule. This is one of the very few proteins found in the human genome which has both the activities in the same gene. The kinase function of LRRK2 has been of utmost importance in PD as it has been observed that enhanced kinase activity of LRRK2 mutants (G2019S, R1441C) leads to neurodegenation and cell death. The kinase domain has reasonale sequence homology to MLK (Mixed Lineage Kinase) subfamily of MAPKKK proteins. How does PD-associated mutant LRRK2 deviate from normal function remains to be elucidated in detail which will in turn help us deduce the normal function of LRRK2 in brains.

alpha-synuclien and LKKR2 in PD

2008-09-19T10:16:00.001-05:00

The two most significant genes which have been widely implicated in familial and sporadic PD are the alpha-synuclein (ASN) and Leucine Rich Repeat Kinase 2 (LRRK2). In PD patients with defects in either of gene (through mutation or other disease causing mechansim), evidence of Lewy Bodies (intracellular esinophillic depositions) and Lewy Neurites are observed in the substantia nigra (SN) of the brain.
PD has been the second most common neurodegenerative disease in the world affecting about 2% of the population. It is a neurological disorder in which degeneration of midbrain dopaminergic neurons leads to loss in motor functions.
I would like to define the cardinal symptons of PD as some one who has been "TRAP"-ed by old age:
T: Tremor
R: Rigidity
A: Akinesia / Bradykinesia
P: Postural Instability

Will discuss more about LRRK2 (the gene of my interest) in subsequent posts, and will evolve a link how GPCRs play a role in PD.