[ZucchiniFlower]

Protecting Neurons from Parkinson's
New insights into the disease's protein culprit
By Katherine Bourzac, SM '04

MIT researchers led by Susan Lindquist, a biology professor and member of the Whitehead Institute for Biomedical Research, have developed a way to protect neurons from degeneration and death in animal studies of Parkinson's disease. The research, which focused on a protein called alpha-synuclein, could lead to therapies for human Parkinson's.

The disease's characteristic tremors and muscle rigidity are caused by damage to and the death of neurons that use the neurotransmitter dopamine to communicate with neighboring neurons. Alpha-synuclein was known to be one of the main causes of that damage; large clumps of it, in a misfolded form, are found in the brains of Parkinson's patients. But researchers did not know what alpha-synuclein's normal role is, why Parkinson's neurons accumulate too much of it, or how it causes disease. Lindquist's team used a yeast model of Parkinson's to study these questions.

Their research suggests that alpha-synuclein plays a role in the process cells use to shuttle proteins between two internal compartments in which critical refinements to proteins are made. Before being shipped off to different parts of the cell, protein strings often need to be cut or folded into three-dimensional shapes, and sometimes groups such as carbohydrates must be added to them.

During these processes, the young proteins are sheltered within protective lipid bubbles. The bubbles also protect the neurons that produce dopamine from damage that can occur if too much dopamine leaks out.

"Dopamine must be packaged in these membranes and sequestered from [the insides of the cell], where it can cause oxidative damage," says Aaron Gitler, a postdoc in Lindquist's lab.

The researchers aren't sure exactly how buildup of misfolded alpha- synuclein disrupts protein trafficking but suspect it disturbs these lipid bubbles. Gitler and Lindquist suggest that as a result, neurons in Parkinson's patients are unprotected from their own dopamine, which thus becomes toxic.

The scientists searched for a way to interfere with this effect. Gene screening showed that activating the gene ypt1, which makes a protein that helps shepherd other, freshly made proteins from one part of the cell to another, did the job: the Parkinson's yeast lived. Rab1, the equivalent shepherding protein in nematode, fly, and rat neurons, also countered alpha-synuclein's toxicity. Rab1 did not completely eliminate neuron death in some of these higher organisms, but it was protective.

Much remains to be done, validation in tests on mice being the most important step. But the Whitehead results have left researchers optimistic about getting at the molecular details of Parkinson's. A complex disease with few treatment options, Parkinson's affects about a million people in the United States. This research represents an important step toward understanding and curing it.

Copyright Technology Review 2006.

[ol'cs]

This paper is a bit hard to digest, but it reminded me of the whole idea of "oxidative damage" of molecules that can be oxidized to form highly reactive intermediates. There has been (as you know) a lot of investigation of why the nigral neurons are dark in coloration. It's because thhey are pigmented by melanin. An intermediate in the polymerization reactions to form melanin is 5,6-Dihydroxyindole, or 5,6-dihydroxyindole -2- carboxylic acid. These molecules are formed from dopamine or dopa by catalytic oxidation reactions. Now the 5,6- dihydroxy part of the molecule is really an "ortho quinone" which is the reactive precursor to melanin formation. Just one little paper of a search on "5,6-dihydroxyindole toxicity" reveals that this molecule in itself is cytotoxic and biochemistry around it may be responsible for cellular damage of dopamine producing cells. The whole idea relates to what you have posted because "dopamine is easily oxidized and must be protected from the rest of the cellular organelles". Thus, it seems that dopamine must be "packaged by heavy duty vesicles" or it leaks out and causes dirty things to happen.
http://www.ncbi.nlm.nih.gov/entrez/q...&dopt=Citation

Is just one paper that outlines the research and thinking along these lines. ONe can do a lot of searching to find good work that may illuminate what is going on in dopamine producing cells. There is an answer yet to all the speculations of the etiology of PD in the research community. I think they are barking up a possible tree of the "how" of PD on this one. There is much chemisry going on that we have hits but no cigar as of yet. cs

[ol'cs]

Best not read if you aren't into technoblurb

In my "old life", I did a lot of research on Alzheimers disease. The current "schtick" about the cause of ALZ still implicates the formation of "plaques and tangles made out of "beta folded sheets" of a 41-42(?) amino acid chain called beta protein, which is just a small peptide cut from a larger transmembrane protein called beta precursor protein. Like alpha synuclein, if it doesn't fold into Beta sheets, it can be eliminated from the body. If it does fold into the very configurationally stable sheets, it can't be eliminated and builds up because, once it forms a sheet, others of its kind are then more eisily "crystallized" epitaxiallly to form the gummed up plaque and tangles that are the undoing of a neuron because this "junk" chokes off neuronal axons and thus kills the cellular functions and the cells die off.
Well, it's a long shot, but serotonergic neurons are present in the cerebral cortex as are a lot of serotonergic axons projecting into the cortex. Serotonin is just 5-hydroxytryptamine, derived from tryptophan (an indole amino acid found in most foods), and, if enzymatically turned into 5,6-dihydroxytrypamine, this is closely related to the Dopamine oxidative cytotoxic 5,6- dihydroxyindole discussed above. You see what i'm getting at? If serotonergic neurons also try to protect 5HT from oxidation and get killed off by producing and using alpha synuclein, then get rid of alpha synuclein protein (a by-product of the vesicles that hold or protect 5-HT neurotransmitter from leaking out into the cytosol where oxidative processes occur) and alpha synuclein is ejected from the cell and misfolds into junk plaques (also found in ALZ patients brains), then the reason why cortical neurons produce beta protein is because the cortical neuron is put in "apoptotic mode" because the serotonergic neurons are dying. Since serotonergic neurons no longer ennervate cortical neurons, then a lot of beta protein is formed as a result of "clipping of the beta precursor protein as part of getting rid of the dying cell.
Thus, protection of serotoneric neurons by stopping the misfolding of alpha synuclein by product, may help protect cortical neurons from dying and clogging up the cortex with plaques and tangles formed by beta protein, which just keeps on forming and killing more cortical neurons. IN essence a cascade effect caused by other junk proteins far from the actual cortical neurons.
I may be dreaming, but that is what science is all about; to think of possibilities and then prove them wrong or right. cs

[ashleyk]

There has been some discussion here on how people with PD lose dopamine producing neurons. And some believe that these neurons have gone dormant.
Inflammation has been increasingly recognized to contribute to the pathogenesis of Parkinson’s disease. Several compounds are neuroprotective at femtomolar concentrations through the inhibition of inflammation. However, the mechanisms mediating femtomolar-acting compounds are poorly understood. Here we show that both gly-gly-phe (GGF), a tri-peptide contained in the dynorphin opioid peptide, and naloxone are neuroprotective at femtomolar concentrations against LPS-induced dopaminergic neurotoxicity through the reduction of microglial activation. Mechanistic studies demonstrated the critical role of NADPH oxidase in the GGF and naloxone inhibition of microglial activation and associated DA neurotoxicity. Pharmacophore analysis of the neuroprotective dynorphin peptides and naloxone revealed common chemical properties (hydrogen bond acceptor, hydrogen bond donor, positive ionizable, hydrophobic) of these femtomolar-acting compounds. These results support a common high-affinity site of action for several femtomolar-acting compounds, where NADPH oxidase is the critical mechanism governing neuroprotection, suggesting a novel avenue of anti-inflammatory and neuroprotective therapy.—Qin, L., Block, M. L., Liu, Y., Bienstock, R. J., Pei, Z., Zhang, W., Wu, X., Wilson, B., Burka, T., Hong, J.-S. Microglial NADPH oxidase is a novel target for femtomolar neuroprotection against oxidative stress.
Parkinson’s disease (PD) is characterized by the specific and progressive death of dopaminergic neurons in the substantia nigra (SN); other neuronal cell types are much less affected. Recent reports have linked inflammation to neurodegenerative disease, where microglia, cells of myeloid lineage responsible for innate immunity in the brain, are considered to be the major cell type underlying the inflammation-mediated neurotoxicity (7 8 9) . The activation of microglia is a complex process involving the release of several soluble proinflammatory factors [tumor necrosis factor {alpha} (TNF-{alpha}), PGE2, IL-1] and free radicals (nitric oxide, superoxide) (7) . Current replacement therapy with L-dopa is able to alleviate disease symptoms, but is unable to alter the disease course. Thus, therapeutic interventions designed to inhibit the microglial inflammatory response offer hope for attenuation of the neurodegenerative disease process. The current anti-inflammatory treatments available, including steroids and nonsteroidal anti-inflammatory drugs, are limited by the ability to influence only a small portion of the microglial response (10) . Thus, identification of compounds acting on novel targets to inhibit the release of a wide range of proinflammatory factors from overactivated microglia is of paramount importance. In the ensuing study, we report that femtomolar concentrations of naloxone and the peptide fragment glycine-glycine-phenylalanine (GGF) attenuate a broad spectrum of the microglia inflammatory response (reactive oxygen species (ROS) and proinflammatory factors) and are neuroprotective with extremely potent efficacy through the inhibition of microglial NADPH oxidase.
http://www.fasebj.org/cgi/content/full/19/6/550

[ashleyk]

There has been some discussion here on how people with PD lose dopamine producing neurons. Some believe that these neurons die off, others believe they have gone dormant. I am not a biochemist as some on this forum are, I find their ideas interesting but hard to follow. I have done internet searches on a drug, naltrexone, which is claimed to be neuroprotective at low doses for many brain diseases such as MS and PD. Most of the research papers I've come across claim that PD and Alzheimers is caused by neuro-inflammation and the neurons die. The researchers work has been with naloxone which they believe is neruo protective. Independantly, a growing number of people with MS believe in low dose naltrexone and they have been taking it over the past few years. Naltrexone is similar to naloxone. I believe there is good reason to look at naloxone/naltrexone, it's possible this drug can stop PD progression and it's available now. I take it.
Ashley

Below is a link to RemedyFind and the people with MS who take LDN.
http://www.remedyfind.com/treatments/21/2165/

Neuropharmacology Section,
{dagger} Laboratory of Structural Biology,
{ddagger} Chemistry Section, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA;
§ Department of Bioscience and Bioengineering, Dalian University of Technology, Dalian, P.R. China; and
|| Department of Neurology, First Clinical Hospital,

"Inflammation has been increasingly recognized to contribute to the pathogenesis of Parkinson’s disease. Several compounds are neuroprotective at femtomolar concentrations through the inhibition of inflammation. However, the mechanisms mediating femtomolar-acting compounds are poorly understood. Here we show that both gly-gly-phe (GGF), a tri-peptide contained in the dynorphin opioid peptide, and naloxone are neuroprotective at femtomolar concentrations against LPS-induced dopaminergic neurotoxicity through the reduction of microglial activation. Mechanistic studies demonstrated the critical role of NADPH oxidase in the GGF and naloxone inhibition of microglial activation and associated DA neurotoxicity. Pharmacophore analysis of the neuroprotective dynorphin peptides and naloxone revealed common chemical properties (hydrogen bond acceptor, hydrogen bond donor, positive ionizable, hydrophobic) of these femtomolar-acting compounds. These results support a common high-affinity site of action for several femtomolar-acting compounds, where NADPH oxidase is the critical mechanism governing neuroprotection, suggesting a novel avenue of anti-inflammatory and neuroprotective therapy.—Qin, L., Block, M. L., Liu, Y., Bienstock, R. J., Pei, Z., Zhang, W., Wu, X., Wilson, B., Burka, T., Hong, J.-S. Microglial NADPH oxidase is a novel target for femtomolar neuroprotection against oxidative stress.
Parkinson’s disease (PD) is characterized by the specific and progressive death of dopaminergic neurons in the substantia nigra (SN); other neuronal cell types are much less affected. Recent reports have linked inflammation to neurodegenerative disease, where microglia, cells of myeloid lineage responsible for innate immunity in the brain, are considered to be the major cell type underlying the inflammation-mediated neurotoxicity (7 8 9) . The activation of microglia is a complex process involving the release of several soluble proinflammatory factors [tumor necrosis factor {alpha} (TNF-{alpha}), PGE2, IL-1] and free radicals (nitric oxide, superoxide) (7) . Current replacement therapy with L-dopa is able to alleviate disease symptoms, but is unable to alter the disease course. Thus, therapeutic interventions designed to inhibit the microglial inflammatory response offer hope for attenuation of the neurodegenerative disease process. The current anti-inflammatory treatments available, including steroids and nonsteroidal anti-inflammatory drugs, are limited by the ability to influence only a small portion of the microglial response (10) . Thus, identification of compounds acting on novel targets to inhibit the release of a wide range of proinflammatory factors from overactivated microglia is of paramount importance. In the ensuing study, we report that femtomolar concentrations of naloxone and the peptide fragment glycine-glycine-phenylalanine (GGF) attenuate a broad spectrum of the microglia inflammatory response (reactive oxygen species (ROS) and proinflammatory factors) and are neuroprotective with extremely potent efficacy through the inhibition of microglial NADPH oxidase.
http://www.fasebj.org/cgi/content/full/19/6/550

[RLSmi]

Ashley, your posts on LDN and the published work by Hong's group are the reasons I began using dextromethorphan, assuming it has the same properties in supressing neuroinflammation as naltrexone. So far, it seems to be working.
As I have said before, one can't be sure if this is a true indication of neuroprotection or simply a sinemet "honeymoon".
In addition to sinemet, amantadine, CoQ10 and DM, I am now taking a tsp of tumeric each day, and plan to increase that to 2 tsp next week. I see no reason to not continue this regimen indefinitely.

[ZucchiniFlower]

Cs, I was able to follow what you were saying but I'm too tired to understand it completely. I'll try again later.

I think that the biochemistry of the brain is hard to reduce to simple theories. There's much more than altered dopamine metabolism going on. Also, something that is harmful to the brain may also in some ways protect the brain. Even turmeric raises acetylcholine, which I'm trying to reduce with my artane.

I thought the following articles are very interesting and illuminate my point:

Pathogenic role of glial cells in Parkinson's disease


Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive loss of the dopaminergic neurons in the substantia nigra pars compacta (SNpc). The loss of these neurons is associated with a glial response composed mainly of activated microglial cells and, to a lesser extent, of reactive astrocytes. This glial response may be the source of trophic factors and can protect against reactive oxygen species and glutamate.

Alternatively, this glial response can also mediate a variety of deleterious events related to the production of pro-oxidant reactive species, and pro-inflammatory prostaglandin and cytokines. We discuss the potential protective and deleterious effects of glial cells in the SNpc of PD and examine how those factors may contribute to the pathogenesis of this disease. © 2002 Movement Disorder Society

http://www3.interscience.wiley.com/c...TRY=1&SRETRY=0

Viewpoint
Challenging conventional wisdom: The etiologic role of dopamine oxidative stress in Parkinson's disease
J. Eric Ahlskog, PhD, MD *

Oxidative stress is well documented in Parkinson's disease (PD) and has been attributed to dopamine oxidative metabolism. However, evidence of oxidative stress is found in a variety of neurodegenerative disorders, suggesting that more general factors are responsible or that cytodestructive processes secondarily generate oxyradical products. Increasing evidence points away from dopamine metabolism as an important contributor to PD neurodegeneration. Predictions from the dopamine oxidative stress hypothesis of PD reveal multiple inconsistencies. Although the clinical and therapeutic importance of the nigrostriatal dopaminergic system is undeniable, PD neuropathology is much more widespread. © 2004 Movement Disorder Society

http://www3.interscience.wiley.com/c...3299/HTMLSTART

Back to the hood (sterile hood, not neighborhood!)

~Zucchini

[ZucchiniFlower]

I don't know how long these posts can be but here's a full article:

Molecular pathogenesis of Parkinson's disease

Sonia Gandhi and Nicholas W. Wood*
Department of Molecular Neuroscience, Institute of Neurology, Queen Square, London

* To whom correspondence should be addressed. Email: n.wood@ion.ucl.ac.uk

Received August 2, 2005; Accepted August 6, 2005


ABSTRACT


Parkinson's disease (PD) is a common and incurable neurodegenerative disease, affecting 1% of the population over the age of 65. Despite a well-described clinical and pathological phenotype, the molecular mechanisms which lead to neurodegeneration remain elusive. However, there is a wealth of evidence from both toxin based models and genetic based models, which suggest a major etiologic role for mitochondrial dysfunction, protein aggregation, the ubiquitin–proteasome system and kinase signalling pathways in the pathogenesis of PD. Ultimately, an understanding of the molecular events which precipitate neurodegeneration in idiopathic PD will enable the development of targeted and effective therapeutic strategies. We review the latest evidence for the proposed molecular processes and discuss their relevance to the pathogenesis of sporadic PD.


INTRODUCTION


Parkinson's disease (PD) is a common neurodegenerative disease first described in 1817 (1). Subsequently, the clinical triad of bradykinesia, tremor and rigidity came to be recognized as core clinical features. One hundred years later, the neuropathological hallmarks underlying the clinical phenotypes were characterized as the loss of dopaminergic neurons in the substantia nigra, together with the presence of intraneuronal inclusions termed Lewy bodies (2). However, despite these early descriptions, the etiology of PD remains unclear. In the last decade, the identification of several genes that cause rare familial forms of PD has revealed novel proteins and pathways that may produce both dopaminergic neuronal degeneration and a clinical parkinsonian syndrome. The genetic burden of actual mutations in the idiopathic or sporadic form of PD is small, accounting for only 5–10% of the overall PD population. However, the strikingly consistent, specific phenotype of familial and sporadic PD has led researchers to believe that one common molecular mechanism may underlie PD. It is hoped that the same pathways underlying familial forms of the disease may have major relevance in the pathogenesis of the sporadic forms. Ultimately, understanding the pathogenesis of the sporadic form of PD will have the greatest impact on advancing novel therapies for this common incurable neurodegenerative disorder.

This review highlights the evidence for the major pathways that precipitate neurodegeneration in PD from genetic analyses, in vitro models of protein function, experimental animal models and postmortem brain studies. We explore the convergence of these pathways and their potential contribution to sporadic PD.


OXIDATIVE STRESS AND MITOCHONDRIAL DYSFUNCTION


The earliest hypothesis of PD pathogenesis was based on the finding that three mitochondrial complex 1 inhibitors, namely MPTP, rotenone or paraquat, were able to reproduce parkinsonism with selective dopaminergic neuronal loss in vitro, as well as in vivo mice (3,4) and primate (5) models. The initial models did not fully reproduce the features of PD, mainly because there was an absence of one of the major hallmarks of PD, the Lewy body. However, a chronic infusion of rotenone in rodents (6) and, more recently reported, a chronic infusion of MPTP in mice (7) have recapitulated the pathological features of PD with alpha-synuclein positive aggregates. This supports the initial theory that sporadic PD may be caused by a combination of environmental toxins acting via inhibition of the mitochondrial respiratory chain to produce selective dopaminergic cell loss and inclusion bodies.

Inhibition of complex 1 has two major consequences: the depletion of ATP, hence impairment of all ATP dependent cellular processes, and the generation of free radicals that cause oxidative stress. There is clear evidence of oxidative stress in postmortem PD brain: elevated levels of lipid peroxidation markers (4-hydroxynonenal and malondialdehyde) and protein nitration have been found in the substantia nigra and Lewy bodies (8). Reduced levels of glutathione and oxidized glutathione, which act as antioxidants, are the earliest marker of nigral cell loss in PD brain (9). Furthermore, a reduction of complex 1 activity by 30% has been described in brain, muscle and platelets of idiopathic PD patients (10,11).

Evidence for oxidative stress is indeed found in many neurodegenerative diseases and it is questionable whether it is truly causal or consequent upon diseased neurons. However, support for a primary role of oxidative stress has emerged from the study of rare familial forms of PD. A variety of missense, truncating, splice site and deletion mutations have been identified in the gene DJ-1 (12), which cause a form of autosomal recessive parkinsonism (Park 7). The exact function of DJ-1 is unclear, but overexpression of DJ-1 appears to protect cells against mitochondrial complex 1 inhibitors and oxidative stress induced by hydrogen peroxide. This effect is abrogated by DJ-1 mutations (13) or by DJ-1 knockdown using siRNA (14). DJ-1 may be able to act directly as an antioxidant because it can be oxidized at the cysteine residue C106. Moreover, it has been demonstrated that endogenous DJ-1 is localised to the mitochondrial matrix and the mitochondrial intermembrane space in addition to its cytoplasmic pool (15). Interestingly, a quantitative proteomic study of the substantia nigra of mice treated with MPTP revealed a significant increase in the protein DJ-1 in mitochondrial fraction of the substantia nigra (16). Together, this evidence suggests that DJ-1 may play an important role in neuroprotection against oxidative stress caused by mitochondrial toxins.

In 2004, missense and truncating mutations within the PINK1 gene were found to cause autosomal recessive PD (17). Bioinformatic analysis reveals that the PINK1 protein consists of a highly conserved kinase domain and a mitochondrial targeting motif. The presence of this N-terminal mitochondrial targeting motif combined with the demonstration by two groups that PINK1 localizes to the mitochondria in transfected cells is noteworthy (18). As with the other genes that cause autosomal recessive parkinsonism, PINK1 has been suggested to have neuroprotective properties against a variety of cellular stresses, a function which is lost by the mutation G309D identified in certain families (17). If future experiments prove definitively that PINK1 has a role within the mitochondria that protects cells from degeneration, this would further strengthen the hypothesis that mitochondria are critically involved in the pathogenesis of PD.


PROTEOTOXIC STRESS: AGGREGATION AND MISHANDLING
TOP
ABSTRACT
INTRODUCTION
OXIDATIVE STRESS AND...
PROTEOTOXIC STRESS: AGGREGATION...
EMERGING PATHWAYS: KINASES IN...
MOLECULAR CONVERGENCE AND...
CONCLUSION
REFERENCES

Many of the neurodegenerative diseases share a common pathogenic process: the abnormal accumulation and processing of mutant or damaged proteins. There are two stages in this event: (1) the process of protein aggregation and (2) the cellular response to abnormal proteins. First, as is seen with several other neurodegenerative diseases, PD pathology is characterized by the tendency of a highly soluble native neuronal protein to progressively polymerize and develop an altered conformation, resulting in intracellular aggregation—a process associated with neuronal dysfunction and loss. In other inclusion body diseases, it remains unclear whether the presence of such inclusions is pathogenic, protective or incidental. Secondly, the abnormal or misfolded proteins are normally targeted via ubiquitination to the proteasome, where they are degraded in an ATP dependent manner. Thus, dysfunction of the ubiquitin–proteasome system (UPS) could lead to the accumulation of cytotoxic damaged proteins, ultimately resulting in neuronal death.

Protein aggregation
The identification of the first familial PD gene as alpha-synuclein (19) led to the important discovery that the major constituent of Lewy bodies in sporadic PD is alpha-synuclein (20). Missense mutations of the alpha-synuclein gene, as well as the increased gene dosage effect of alpha-synuclein gene triplication (21), cause autosomal dominant familial PD. The human pathology associated with these mutations reflects a widespread and fulminant disease process with nigral cell loss, alpha-synuclein positive Lewy bodies in the brainstem, cortical Lewy bodies (22) and glial cell inclusions (23).

In its native state, alpha-synuclein is a soluble and unfolded protein. Owing to a central hydrophobic region in the protein, alpha-synuclein has a high propensity to aggregate and initially forms an intermediate annular structure called an oligomer or protofibril and ultimately forms insoluble polymers or fibrils (24). These insoluble fibrils are the major constituent of Lewy bodies. It is unclear whether the fibril, the protofibril or the soluble species is the most toxic species in neurons. A variety of factors promote the aggregation of alpha-synuclein: mutations promote the formation of the oligomeric, but not the fibrillar, species (25); posttranslational covalent modifications of the protein such as phosphorylation, nitration and glycosylation may contribute to aggregation (26); and dopamine itself is able to stabilize the alpha-synuclein protofibril by forming a dopamine–alpha-synuclein adduct (27).

Reproducing the features of PD in experimental animal models has met with mixed success: there is no significant loss of neurons in the substantia nigra in transgenic mouse models (28). In contrast, viral mediated overexpression of alpha-synuclein induces nigral degeneration in rats (29). Similarly, Drosophila models based on the expression of normal and mutant forms of human alpha-synuclein show selective loss of dopaminergic neurons and the formation of alpha-synuclein inclusions (30). Moreover, this model has confirmed that phosphorylation at the Ser129 residue is crucial to the toxicity of alpha-synuclein and mutations of this serine residue, which prevent phosphorylation, also abolish the toxicity (31). Interestingly, the reduction of toxicity in this model is associated with increased inclusion body formation, suggesting that inclusion bodies may protect neurons by reducing the amount of diffuse toxic protein by sequestrating it in inert bodies.

Ubiquitin–proteasome system
The first evidence of a direct role of the UPS in neurodegeneration emerged after the identification of the parkin gene. Mutations in the parkin gene are known to cause a large proportion of early onset autosomal recessive parkinsonism (32). There is a wide spectrum of parkin mutations ranging from large homozygous deletions to multiplications, small deletions/insertions and missense mutations. Pathologically, parkin mutations are associated with significant dopaminergic neuronal loss in the substantia nigra and the locus coeruleus. However, in the few parkin related PD cases that have come to autopsy, there is a notable absence of Lewy bodies in patients with the homozygous deletions of parkin, although Lewy bodies are present in patients with compound heterozygous parkin mutations (reviewed in 33). These findings suggest that parkin may play a significant role in Lewy body formation, but conversely, nigral cell loss and clinical parkinsonism can occur in the absence of inclusion body pathology.

Parkin encodes an E3 ubiquitin ligase with the characteristic two RING (really interesting new gene) finger domains separated by an IBR (in-between ring) domain that is common to other E3 ligases (33). These enzymes catalyze the addition of ubiquitin chains to target proteins before its destruction by the proteasome. Many putative parkin substrates have been identified including synphilin-1, O-glycosylated alpha-synuclein, Pael-R, CHIP, cdc-Rel1A, cyclin E, synaptotagmin X1 (reviewed in 35). Loss of parkin function may lead to accumulation of its substrates, which would ultimately lead to neuronal cell death. Indeed, overexpression of the parkin substrate Pael-R produces dopaminergic cell death in vitro, which can be rescued by parkin overexpression (36).

Despite these in vitro findings, modelling parkin associated PD in vivo has proved challenging: mice with targeted deletion of exon 3 of parkin do not show nigral neuronal loss (37). The parkin knockout model in Drosophila clinically shows locomotor dysfunction due to peripheral muscle degeneration rather than dopaminergic neuronal loss (38).

Further support for the role of the UPS was provided by the identification of UCHL-1, another gene implicated in causing dominant PD (39). UCHL1 is a ubiquitin C-terminal hydrolase L1, which aids the recycling of polyubiquitin chains back to monomeric ubiquitin. The genetic evidence for UCHL-1 is less strong than for genes such as parkin, because the initially identified mutation has only been demonstrated in two siblings with PD and has not been reported in any additional families. A mouse model with an inframe deletion of exons 7 and 8 of UCHL-1 demonstrates gracile axonal dystrophy, sensory and motor ataxia with accumulation of beta amyloid and ubiquitin deposits, but without evidence of nigrostriatal neuronal loss (40).

There is growing evidence that the UPS may be important in the pathogenesis of sporadic PD. Postmortem brain tissue from patients with idiopathic PD show functional deficits in the 20S proteasome activity (41). Administration of synthetic and natural inhibitors of the UPS to rodents for 2 weeks produces selective nigral cell loss and Lewy body-like inclusions, together with clinical signs of bradykinesia, rigidity and tremor (42). Thus, a primary aberration in the UPS induced in vivo (as described earlier for complex 1 inhibitors) is able to reproduce many of the specific features of PD.


EMERGING PATHWAYS: KINASES IN PD


The discovery of the PINK1 gene focused on the potential role of kinases in the neurodegenerative process. Kinases are known to have major roles in cell cycle signalling and are the most common domain encoded by cancer genes. PINK1 encodes a serine/threonine kinase with significant homology to the calcium-calmodulin protein kinases. PINK1 was initially identified as a kinase that was upregulated on overexpression of PTEN, a tumour-suppressor gene, suggesting that PINK1 may play a role in cell cycle regulation (43). Moreover, transient knockdown of PINK1 renders cells susceptible to apoptosis on exposure to taxol (44). Thus, the neuroprotective function of PINK1 may actually lie in the direct regulation of a programmed cell death pathway, occurring as a much later event than either oxidative stress or UPS dysfunction.

The subsequent identification of the LRRK2 gene as the Park 8 locus added further interest in the importance of kinases in mediating neurodegeneration (45,46). Missense mutations in this large gene were found to cause autosomal dominant PD in pedigrees from Basque and the UK. LRRK2 mutations cause a range of differing pathologies including neuronal loss in the substantia nigra either in the absence of Lewy bodies or in the presence of widespread Lewy body disease or in the presence of neurofibrillary tangles. The predicted product of the LRRK2 gene is a 286 kDa protein called dardarin: dardarin is a member of a novel family of protein kinases which have sequence similarity to both tyrosine and serine/threonine kinases. In addition to the kinase domain, there are several other conserved domains such as the leucine-rich repeats, the WD40 domain and a Ras/small GTPase superfamily domain (47). As yet, little is known about the function of LRRK2, although the presence of these novel domains suggests a unique function in dopaminergic survival and perhaps a hitherto unknown pathway that leads to nigral cell loss.


MOLECULAR CONVERGENCE AND DIVERGENCE


The inherited forms of parkinsonism (Table 1) demonstrate how a single molecular aberration is sufficient to independently reproduce the clinical and pathological features of PD. Moreover, the mutation is present in all cells of the body, and yet, the cell loss is restricted to the substantia nigra and associated structures, informing that these molecular events must be cell specific to precipitating PD-type neurodegeneration. However, with the growing wealth of genetic clues, comes increasingly complex puzzles: how can one reconcile the fact that the PD phenotype can occur in the absence of Lewy bodies (for example, in LRRK2 and parkin-associated parkinsonism) and yet a biochemical excess of normal alpha-synuclein as a result of alpha-synuclein gene triplication is also sufficient to cause PD? Is it possible to place the UPS, mitochondrial function, oxidative stress, protein aggregation and kinase signalling in one unified pathway that leads to nigral cell death? Furthermore, if there are instead several distinct pathways that are separately able to precipitate nigral cell death, which of these is the most significant in sporadic PD?




Table 1. Summary of the genes accounting for the Mendelian forms of PD and the major published mutations identified in these genes

If all the described cellular processes (Figure 1) do have relevance in sporadic PD, then one would expect to find evidence of molecular convergence between them. Certainly, the mitochondrial system and the UPS do not exist separately: complex 1 inhibitors cause a reduction in proteasomal activity (47), and conversely, proteasomal inhibitors can cause mitochondrial damage (49). Inhibiting proteasome function renders cells more susceptible to oxidative stress through complex 1 inhibition (48).

In addition to its defined role in the UPS, parkin plays a less well understood role in the mitochondria: for example, the most prominent feature of the parkin knockout in Drosophila is mitochondrial pathology in the flight muscles, resulting in apoptosis. Furthermore, a proteomics approach in the parkin knockout mouse revealed that the most notable changes were in mitochondrial proteins in the electron transport chain, an event associated with a reduction in mitochondrial respiratory capacity (50). There is also evidence of a relationship between mitochondrial dysfunction and protein aggregation: complex 1 inhibition and other forms of oxidative stress lead to alpha-synuclein aggregation (51).

Moreover, mice lacking the alpha-synuclein gene are resistant to the toxic effects of MPTP, suggesting that dopaminergic neuronal degeneration requires both mitochondrial dysfunction and subsequent alpha-synuclein aggregation to occur (52). In turn, aggregated alpha-synuclein is able to inhibit the proteasome and thus interfere with the UPS (53,54). Thus, there are multiple levels of intersection between these pathways such that a relatively minor abnormality in one or more cellular processes can be amplified by its interaction with other cellular processes to potentially result in several forms of stress (proteasomal, oxidative and aggregation) that would ultimately force a cell into programmed cell death.



Figure 1. Molecular mechanisms leading to neuronal cell death. The grey boxes encompass the independent pathways that can lead to cell death, i.e. mitochondrial dysfunction, ubiquitin–proteasomal dysfunction and alpha-synuclein aggregation. The red circles highlight the primary aberrations that affect each pathway to trigger cell death, such as mutations in the Mendelian genes, or administration of toxins in animal models. The blue arrows indicate the convergence of these pathways: impairment of the UPS has adverse effects on mitochondrial function and the generation of ROS, and similarly, oxidative stress alters the function of the UPS. Oxidative stress may increase alpha-synuclein aggregation, and aggregated alpha-synuclein inhibits the function of the UPS. Note that it is not yet known how LRRK2 mutations cause nigral neuronal loss in PD.


Sporadic PD is a late-onset neurodegenerative disease that has been traditionally thought to be due to exposure to an environmental toxin on the background of a genetically susceptible individual. In this model, it could be conceivable that genetic variation in genes encoding proteins along several pathways, such as the UPS, the mitochondrial respiratory chain or alpha-synuclein handling, would predispose individuals to low levels of chronic, relative dysfunction of these molecular processes. There is growing evidence from genetic association studies that genetic variation in such genes may contribute as susceptibility factors in sporadic PD (55,56). Cumulative ‘normal’ stress over a long period of time, such as the ageing process (which is known to be associated with reduction in the UPS function and increased levels of oxidative stress), or exposure to toxins, could then tip the balance from a genetically determined stressed cellular state to programmed cell death in that particular individual. This explanation would unite all the molecular pathways implicated in Mendelian forms of PD into a complex multifactorial model for sporadic PD.

CONCLUSION


The themes of protein aggregation, mitochondrial dysfunction and proteasomal stress continue to recur in many of our models, and therefore, may play a central role at some stage of PD pathogenesis. However, there remains a host of questions in this field that need to be addressed. For example, primary events must be distinguished from secondary consequences of stressed diseased neurons. Defining prerequisite events from contributory events in the PD pathway may eventually yield potential therapeutic targets. A clearer understanding of exactly how and when these pathways overlap and converge to produce nigral neuronal degeneration will be vital to understanding the pathogenesis of sporadic PD.

To date, neither genetic studies nor postmortem brain studies are able to inform on the temporal sequence and relationship of the various cellular processes. These questions may only be convincingly answered by in vivo models, although hitherto no single toxin or gene-based model has fully recapitulated the pathological and clinical features that define human PD so clearly.

Perhaps, we will finally conclude that sporadic PD encompasses a heterogenous spectrum of disease, whereby several distinct molecular mechanisms may converge to produce a final common pathological and clinical phenotype in different individuals, a model that may have implications for our understanding of the aetiology of all neurodegenerative diseases.

http://hmg.oxfordjournals.org/cgi/co...ull/14/18/2749

[ol'cs]

You sure hit the nail on the head when you said how difficult it is to make any sense out of PD research. The biochemical mechanisms that control the syndrome are "WAY OUT THERE". Not something as simple as a virus that causes a certain disease, something that can be pinned down and logic applied to countering it's action (like reverse transcriptase of RNA did so much to halt AIDS in it's tracks).
The causes of PD are a biochemical universe unto itself. Nothing is simple. Most of whats out there (a virtual library of Alexandria) concerning PD etiology is "grasping at straws" and "hand waving". No doubt, there IS an answer, but that answer can't be distilled into anything less than thousands of possibilities. The scope of "our" disease is devilishly frustrating to all those who attempt to explain the various specific aspects of PD. What should be "simple things" like why some of us shake like leaves in the wind, or some of us being rock solid are at present just impossible to tease out from the overall syndrome. I've perused so many research papers that take forever to get through because i have to hit the books at every corner to learn the science that i'm not familiar with; and then dream what Einstein called "thought experiments" that more often turn out to be exactly that, more silly dream than logic. This disease is still going to take decades to figure out, but figure it out we must, because that's the first step to finding a cure. Answering pivotal questions such as "will stem cell research work"; is there any hope to small molecule therapy"; is PD a response to toxins (endogenous or exogenous); are genetic mutations involved; is it related to lifestyle preferences; is it a lack of neccessary enzymes or their cofactors that lead to the syndrome? WHAT? WHAT causes PD. There are so many trees to bark up at. WE know some things, and can apply Rumsfeld logic to the things we know, the things we don't know, and the things we know we don't know, but to date, this has still not bourne fruit.
Well, I'll just keep my mouth shut untill i can read your latest contributions and can reply with more than the fluff that I have in this post.
Keep on reading!!! It's the only way to get step one over with. Remember, all of you out there who have some form of PD; researchers can find things that they can speculate to work for us, but it is ONLY US who can tell them for sure if they are on the right track.
At this point, i consider myself dispensable, and would be the first to swallow any new pill, be injected with any substance, and be a big white rat for any idea that I believed in, if i thought that it would really benefit those who have yet to travel this road. And lets not forget that right now , there are many many "soldiers in the war against PD" who have already given of themselves just to get us where we are today (you know who you are). This is a disease on the scale and effects of such things as cancer, AIDS, MS, Lou Gehrigs and a whole spectrum of diseases that are the bane and the misery of mankind. IT's sufferer's deserve at least the best that the best can do for them. Amen! cs

[ZucchiniFlower]

Cs, thanks for your sweet post. I enjoyed your Rumsfeld reference.

It will take much time to figure PD out, but I'm grateful that symptomatic relief is better than it was when my dad had PD. I'm glad your apomorphine is helping you!

If I could really slow progression, and also relieve many of my symptoms, I'd be a happy camper, even without complete answers to our questions.

I'm short on time now. Working overtime today so I can take a few days off. My mother is arriving tomorrow. Luckily, at 86 she's in much better shape than me.

Happy Thanksgiving, everybody. Hope you are all well enough to enjoy a good meal, and hopefully good company.

~Zucchini