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.
Thursday, October 22, 2009
Wednesday, October 21, 2009
Therapeutic modification of alpha-synuclein
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.
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.
Monday, October 19, 2009
In Vivo models of alpha-synuclein
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.
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
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.
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