Introduction

Parkinson's disease (PD) is one of the most common neurological disorders, second only to Alzheimer's disease, affecting approximately 2% of adults. Although the cause of the idiopathic form of the disease is yet to be established, recent advances in the study of certain familial forms of the disease have shed some light on the overall process of neurodegeneration in PD. Still, much remains to be understood about the disease. The recent development of new techniques such as the use of short RNA molecules like small interfering RNAs (siRNA) have gained increasing popularity as tools to knockdown certain cellular mRNAs, and these tools have been valuable in elucidating molecular pathways and players involved in PD. Still, the ultimate goal is to treat the disease, and the current understanding of the potential etiology of PD makes it an attractive target for a siRNA therapy. One common feature amongst the various forms of the disease is protein accumulation and aberrant protein clearance. Hence, a reduction in translation of specific proteins may suffice to relieve the cellular stresses. In addition, a number of cell death signaling events likely to be involved in all forms of PD are also potential siRNA targets.

RNA interference

RNAi is a response to double-stranded RNA that has been identified in numerous organisms in all three eukaryotic kingdoms. The major enzymes involved are highly conserved amongst species, indicating that the response is an important cellular mechanism. Work into the world of naturally occurring siRNA has shown that this response is not only involved in protecting the genome from threats such as viruses,¹ but is also mechanistically related to microRNA (miRNA) pathways that provide crucial regulation of temporal control of development. The understanding of the mechanisms involved in RNA interference has progressed dramatically in the past several years. Briefly, long double-stranded RNA are processed by an RNase III enzyme named dicer to shorter (21–23 nt) siRNA complexes. The actual gene-silencing is processed by a group of proteins collectively termed the RISC (RNA-induced silencing complex), which serves to bring the siRNA to the vicinity of a complimentary mRNA and provides the catalytic enzymes involved in the cleavage of the mRNA.^{2, 3, 4} miRNA works along a similar pathway, however, naturally occurring miRNA precursors are short (70 bp) non-coding hairpin sequences that are also processed by dicer into short stretches of single-stranded RNA. Unlike siRNAs, miRNAs contain short mismatches in the sequences complementary to their target mRNAs, and miRNAs are thought to inhibit translation rather than lead to the degradation of mRNA. miRNAs are extremely important in gene regulation, and certain neuronal disorders such as fragile X are linked to the miRNA pathway.⁵

The siRNA system has been commandeered to create a valuable tool in order to study the effects of knocking down certain transcripts. Experimentally, one scans a target gene for suitable target sequences 21–23 nt long and designs double-stranded RNAs to target these sites. Scanning is typically assisted by one of several public-access computer programs that incorporate experimental 'rules' for designing effective siRNAs. These rules include avoiding runs of four or more guanosines or adenosines, keeping the base composition between 30 and 55% G+C, and identifying sites with asymmetric thermal stability so that the antisense strand is preferentially incorporated into RISC.^{6, 7, 8, 9} As an alternative for rational design of siRNAs, several groups have developed strategies for creating and screening siRNA libraries targeted to a gene of interest.^{10, 11, 12, 13} This approach is valid because some of the most effective siRNAs that have been described in the recent literature would have been avoided according to the current design algorithms.

Delivery of siRNA

Short interfering RNAs can be introduced to mammalian cells and experimental animals either as RNA or as DNA clones that code for the double-stranded RNA. When DNA clones are used, they are often designed to produce short hairpin RNA (shRNA) that is processed to form siRNA in the cell. For cultured cells and for temporary dosing to tissue, direct delivery of siRNA has distinct advantages: short RNA molecules are commercially available in pure form. Double-stranded RNA can be stabilized by chemical modifications in both strands of the duplex.^{14, 15, 16} Certain ribose modifications in the sense strand have the added advantage of increasing the incorporation of the antisense strand of the duplex into RISC, therefore increasing cleavage of the intended mRNA. Using RNA also permits accurate dosing of the cells, since a known quantity of RNA is added. The disadvantage of direct RNA treatment is that a large amount of siRNA is required (typically 10–100 nM) and the effect is temporary (3–4 cell generations). Using a high dose of siRNA may saturate the nuclear-cytoplasmic transport of miRNAs and thus lead to non-specific effects.

Plasmids and viral vectors can be used to deliver shRNAs, which are usually produced under the control of RNA polymerase III (pol III) promoters. Pol III promoters generate large amounts of transcript, but much of it is sequestered in the nucleus away from the cytoplasmic RISC. Consequently, some investigators have switched to pol II promoter systems for the expression of shRNA¹⁷ or have disguised their siRNA hairpins as miRNA precursors to facilitate transport to the cytoplasm.¹⁸ Delivery with viral vectors has the advantages that hard-to-transfect primary cells can often be infected and that expression of the RNA hairpin may remain stable in nondividing cells or for many generations in dividing cells, if an integrating virus is employed.

While the delivery options are plentiful in the laboratory, bringing siRNA to the clinic reduces these options. Obvious problems arise from RNA stability as well as access to target organs. Nonviral delivery methods to the mammalian brain have included both non-specific lipid and polyethylenimine RNA complexes and cell-specific methods. For example, receptor-specific pegylated immunoliposomes (PIL) have proved successful when injected directly into tissue. PILs are 'nano-containers' with the ability to store plasmid DNA and are heavily coated with 2000-Da polyethyleneglycol (PEG). Tethered to a fraction of the PEG are antibodies specific for those cell types to be targeted. For instance, in a mouse model for intracranial human brain cancer, plasmid-encoded siRNA-targeting human epidermal growth factor receptor was injected with this vehicle, significantly increasing the survival of the animal.¹⁹ As an alternative to siRNA lipid complexes, electroporation of 'naked' RNA has been shown to be effective in delivering siRNA to a limited number of target organs, including the visual cortex and the CA1 region of the hippocampus.²⁰ As with lipid complexes and RNA-nanoparticles, however, electroporation leads to only a transient reduction of gene expression, and the prospect of sequential re-administration to the brain is unattractive in a therapeutic setting.

Viral vectors provide the most suitable means of delivering and expressing siRNA for long-term therapy. Several recombinant viruses have been used for gene therapy in the brain, but the short duration of transduction and the proinflammatory properties of vectors based on adenovirus and herpes simplex virus have restricted their use to antitumor applications. Lentivirus vectors and recombinant adeno-associated virus (rAAV) vectors have been used for long-term transduction of the brain and particularly for gene therapy in animal models of neurodegenerative disorders such as Huntington's disease (HD),^{21, 22} spinocerebellar ataxia²³ and PD.^{24, 25} Both virus types can infect nondividing cells and lead to long-term gene expression. Lentiviral vectors give a more localized response and can contain a larger 'payload' in terms of passenger genes, but delivery of siRNA does not exceed the 4.7 kb carrying capacity of AAV. While rAAV does not provoke an inflammatory response, infection does stimulate humoral immunity, and most people have circulating antibodies to AAV.^{26, 27} Indeed, pre-immunization of rats with AAV2 prevents striatal transduction with rAAV2 vectors.²⁸ Several rAAV serotypes have been used to deliver marker genes or therapeutic genes to the midbrain, but to date, rAAV1 and rAAV5 appear to provide the widest area of transduction, while gene expression mediated by rAAV2 infection is more precisely located near the site of injection.²⁹ siRNA therapeutics will almost certainly need to specifically target the SN. rAAV2 has the serendipitous feature that it transduces the nigral DA neurons with higher efficiency than other neurons in the anatomical vicinity providing specific nigrostriatal transduction.^{29, 30, 31}

RNA interference holds a valuable potential as a future therapeutic, it could be used to simply knockdown a specific disease gene. siRNA is selective for fully complementary mRNA targets, and short mismatches has been shown to significantly lower the efficacy of target mRNA degradation.³² Nevertheless, as few as 11 sequential matches between the 3' end of the target RNA and the 5' end of the antisense strand are sufficient to mediate cleavage by RISC, so that allele specificity is thought to require three or more mismatches with nontarget RNAs. Allele specificity would prove valuable as it would enable treatment of dominant mutations by targeting only the mutant sequence. If specificity could not be achieved by targeting the degradation of an RNA bearing a point mutation, RNA replacement might be achieved by concomitantly expressing a 'hardened' mRNA, in which several silent nucleotide substitutions have been introduced. Thus, this approach essentially replaces the defective gene with a healthy one.

Recent work on an animal model for spinocerebellar ataxia type 1, a polyglutamine expansion disease characterized by progressive neurodegeneration, demonstrates the potential for pre-translational silencing of a gene.²³ Polyglutamine diseases are characterized by the presence of intracellular inclusions containing the expanded protein as well as numerous components of the machinery involved in ubiquitin-mediated proteasomal processing. It has been shown in vitro that lengthier repeats cannot be processed through ubiquitin-mediated proteasomal degradation. These uncleaved stretches of amino acids are thought to further inhibit the proteasome and are also very prone to aggregation which may be toxic to the cell.³³ However, when Davidson et al.²³ injected a rAAV expressing a siRNA targeting the ataxin-1 mRNA into the cerebellum, they demonstrated that transduced cells had a lower level of inclusions and that the animals displayed a significant improvement in motor performance. Similar positive results were obtained delivering shRNAs to huntingtin in polyglutamine-expanded HD mice.^{21, 22}

Similar to spino-cerebellar ataxia, several other neurodegenerative diseases are also characterized by the accumulation and aggregation of protein. In certain familial forms of Amyotrophic Lateral Sclerosis, a mutation in the gene encoding the antioxidant enzyme Cu,Zn superoxide dismutase, is thought to cause protein misfolding, oligomerization and/or proteasome inhibition.^{34, 35} siRNA treatment in transgenic animals carrying these mutations targeting the proapoptotic gene prostate apoptosis response-4, a protein shown to be upregulated in the lumbar spinal cord samples of ALS patients, protected from caspase 3-mediated apoptosis and preserved mitochondrial function in a staurosporine regimen.³⁶ RNAi treatment has shown some promise in transmissible spongiform encephalopathies where the transmittable conformational change of an aberrant isoform of the prion protein causes neuronal cell death. When transfected into a scrapie-infected neuroblastoma cell line, siRNA targeting the prion mRNA reduced the levels of the protease-resistant isoform of the protein.³⁷

In PD, several familial forms have been identified and linked to disease genes, several of which are thought to be inhibitory to proteasomal processing, or prone to accumulation and aggregation. It is also likely that this common theme of protein accumulation and aberrant protein degradation also share common downstream effectors, such as common cell death triggers which also become attractive targets in a siRNA-based therapy (Figure 2).

Figure 2.

Schematic of potential etiological factors of PD and potential siRNA targets (red symbols). Accumulated -synuclein is a common feature in PD, and as such it serves as a potential target for pre-translational silencing (1). In parkin-associated disease, a number of substrates are thought to accumulate, some may be detrimental due to function (e.g. cyclin E) (2), or simply due to accumulation (3). The newly identified genes (DJ-1 and PINK) may be involved in protection against oxidative stresses, which may be induced by endogenous factors such as by-products of DA metabolism, or exogenous factors such as environmental toxins impairing mitochondrial function. Increases in oxidative stress also acts as to create a general increase in misfolded proteins, thus potentially triggering apoptosis directly through an unfolded protein response (3, 4), or indirectly through mitochondrial signaling events (4). Finally, since apoptosis is suspected as the final common pathway in DA neuron cell death, blockade of proapoptotic proteins in neurons late in the apoptotic cascade may be therapeutic (5).

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Parkinson's disease

The average age of onset of the idiopathic form of the disease is at the age of 60 years, and the disease symptoms usually appear on one side of the body with a subsequent bilateral progression. Noticeable motor dysfunctions include a highly characteristic resting tremor, rigidity, bradykinesia, loss of postural reflexes and a distinguishable freezing phenomenon.³⁸ Diagnosis cannot be complete without establishing a responsiveness to levodopa, a neurotransmitter replacement strategy, and currently the standard pharmacotherapy for PD.³⁹ This treatment is limited, however, by the appearance of side-effects including reduced duration of therapeutic response to a single dose and peak-dose dyskinesias 3–5 years after the commencement of therapy.⁴⁰ There are numerous experimental treatments that are currently undergoing clinical evaluation.^{24, 41, 42} Nonetheless, there is no protective treatment or a cure for PD.

Neuropathologically, the disease is defined as a progressive depletion of striatal dopamine (DA) with the concomitant loss of the dopaminergic neurons of the substantia nigra pars compacta (SN). Similar to the polyglutamine repeat disorders mentioned previously, a hallmark feature of PD is the presence of intracytoplasmic inclusions, termed Lewy Bodies, which are highly immunoreactive to components of the proteasomal machinery such as ubiquitin and proteasomal subunits.⁴³

The cause of the idiopathic form of the disease is still unknown and is probably multifactorial. Numerous hypotheses have been put forth claiming that environmental factors such as pesticides or metals may be exogenous factors in the disease.^{44, 45} However, it appears likely that subsequent steps in the disease process include mitochondrial impairment and increased oxidative stress.⁴⁶ At the point in time when motor functions are impaired, approximately 80% of striatal DA is already gone, and the actual mechanism leading to cell death in the idiopathic form of the disease is still unknown. Therefore, the need for a more complete understanding of the disease progression is obvious. Additional clues to the disease etiology have come with the discovery of familial forms of PD and the genes involved in these forms.

-synuclein

The first gene to be linked to PD was PARK1 that was identified in 1997 by Polymeropoulos et al.⁴⁷ by studying several families in southern Europe. The mutation identified in the -synuclein gene was a single point mutation (A53T), and subsequently, several other mutations were also identified. In addition, a gene triplication was discovered in a US family,⁴⁸ suggesting that overexpression of the gene is detrimental as well. -synuclein-familial PD differs from the sporadic disease in that it is generally earlier in onset, and has a greater component of dementia. The parkinsonism is often considered a subset in a more general disorder termed diffuse Lewy body disease. The function of -synuclein is still unknown, although there are many indications that it is involved in the maintenance of synaptic DA vesicles and pre-synaptic function.

-synuclein is considered a natively unfolded protein with a high propensity to aggregate, with the end product being a highly insoluble polymer termed fibrils. It is still unclear which is the toxic event involved in -synuclein-associated disease, but what all mutations have in common is that they enhance the formation of an oligomer intermediate (protofibril) to fibril formation, and this protofibril is thought to possibly have the ability to damage cellular membranes.⁴⁹

-synuclein is a major component of LBs, the presence of misfolded -synuclein and proteasomal machinery led to the idea that LBs are, in fact, neuroprotective, a way for the cell to harness damaging misfolded, insoluble proteins into a contained 'compartment'.

The -synuclein deficient mouse is relatively absent of pathology and also experiences an attenuated response to certain neurotoxic stimuli such as a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) regimen, a common model toxin for nigro-striatal degeneration. The MPTP metabolite 1-methyl-4-phenylpyridinium (MPP⁺) is inhibitory to mitochondria leading to decrease in respiration and increase in the production of oxidative free radicals, subsequently leading to cell death through apoptosis or necrosis.⁵⁰ Considering the tendency for -synuclein to aggregate the animal model data, and the toxicity involved in overexpression, targeting this gene with siRNA could hold therapeutic potential.

Parkin

The PARK2 gene was identified in 1998 to be involved in an early onset form of PD; autosomal recessive juvenile PD (AR-JP).⁵¹ A distinguishable feature of this form of PD is that onset happens as early as in the teens. However, following the identification of this gene, several researchers identified PARK2 polymorphisms as a potential risk-factor in sporadic PD.⁵² Interestingly, patients with this form of the disease lack LBs, suggesting that parkin is required for their formation/maintenance. The gene product parkin is an E3 ligase involved in targeting certain substrate proteins for degradation by the proteasome.⁵³ Briefly, ubiquitin is activated through an ATP-dependent process by an E1 ubiquitin-activating enzyme. The activated ubiquitin is then transferred to an E2 ubiquitin-conjugating enzyme. E2 enzymes act in combination with E3 accessory subunits that bind to specific degradation signals in protein substrates.

Parkin has two RING (really interesting new gene) domains in the carboxy-terminus of the protein which serves as a binding region for the E2. There is a unique parkin domain adjacent to the ring domains which is thought to serve as a substrate recognition region. Thus, parkin serves to facilitate the transfer of activated ubiquitin from the E2 to the substrate creating a substrate 'tagged' with a poly-ubiquitin chain, thereby targeting it for degradation in the 26S proteasome. The substrates that have been identified for parkin are CDC-rel 1 and CDC-rel 2 (septin family, associated with synaptic vesicles), cyclin E, Pael-Receptor (G protein-coupled receptor), p38 tRNA synthase, synaptotagmin XI, synphillin-1, as well as parkin itself.⁴⁹ The cyclin E substrate is unique in that parkin is part of a multiprotein complex which includes Hsel-10 and Cullin-1.⁵⁴ siRNA was used to evaluate the toxicity of some of these substrates. In Drosophila, Pael-R overexpression causes a selective degeneration of dopaminergic neurons, a condition that can be repressed through coexpression of a fly homologue of parkin (dparkin). However, when expressing a siRNA designed against dparkin, the Pael-R mediated degradation is accelerated, indicating that the endogenous dparkin is involved in the degradation of Pael-R.⁵⁵ The inverse experiment would obviously be a natural extension of this test; using siRNA to target Pael-R in a parkin-deficient animal should inhibit cell death due to Pael-R accumulation. However, parkin knockout mice display an incomplete pathology with little connection to the human disease, and no accumulation of substrates.⁵⁶

Similarly, Staropoli and co-workers used a siRNA against parkin to study the effects on cyclin E accumulation. Cyclin E is involved in the regulation of G1/S cell-cycle transition, and when upregulated in mitotic cells would lead to cell division, but in post-mitotic neurons increased cyclin activity is believed to trigger apoptosis.⁵⁷ Cyclins have been shown to accumulate in response to proapoptotic stimuli such the excitotoxin, kainate. Furthermore, cyclin E has been shown to accumulate in parkin-deficient cells and tissue, and more importantly, in the brains of AR-PD patients. Parkin overexpression in cerebellar granule cells was shown to relieve toxicity and apoptosis induced by kainite. However, siRNA-mediated parkin reduction was shown to sensitize the cells to, and enhanced the effects of, kainite toxicity.⁵⁴

DJ-1 and PINK-1

Additional early onset familial forms of PD were discovered recently with the identification of two disease genes involved in mitochondrial function. The first one to be identified was the gene encoding DJ-1.⁵⁸ The function of this gene remains unknown, and numerous hypotheses have been put forth. Probably, the most interesting proposed function in terms of PD pathology is the suggested role of DJ-1 in improved protection against oxidative stresses, perhaps acting as a cellular sensor for oxidative stress and modulator of gene expression.⁴⁹ This was demonstrated in vitro when a siRNA targeting DJ-1 was expressed in the SH-SY5Y neuroblastoma cell line. These cells became more susceptible to the MPP+ and 6-OHDA toxins, similar to cells overexpressing the disease mutation.⁵⁹

The second new familial PD gene identified was PINK-1, a mitochondrial kinase, which was shown to protect cells against proteolytic stresses, although it is unsure by which mechanism this protection occurs.⁶⁰

UCHL1

An additional gene that has been linked to PD is PARK5 coding for ubiquitin C-terminal hydroxylase L1 (UCHL1).⁶¹ As the name suggests, this protein is also involved in ubiquitin-dependent protein degradation by acting as a deubiquitination enzyme removing ubiquitin from processed amino-acid fragments, and, as such, fits with the general idea of impaired protein degradation. However, only a single family has ever been identified with this gene allele, and more work needs to be done to assess its relevance to PD.

The genetic linkages that have been made in PD provide valuable insight and clues to the pathology involved in familial forms as well as in sporadic disease; after identifying the -synuclein form of the disease the field soon realized that -synuclein may also play a role in idiopathic disease. The identification of parkin mutations and the lack of LBs hints to the idea that proteasomal processing play an integral part of the molecular pathology, and the DJ-1 and PINK mutations also involves the mitochondria and oxidative stress in the disease. Although these are distinct molecular processes, they are likely to be interconnected and not necessarily exclusive. Given these ideas it is also likely that the end result of these various forms of the disease converge on a common path of cell death. It is inherently difficult to study cell death in the human PD brain, given the slow rate of cellular death occurring over an extended period of time (decades).

Even so, there are numerous reports of TUNEL-positive nigral neurons from patients with PD, suggesting that cell death is mediated through apoptosis, which may be a consequence of mitochondrial failure and oxidative stress. Further support for this idea comes from reports that show that dopaminergic neurons from PD brains show an upregulation of proapoptotic genes, and the fraction of neurons containing activated caspase 3 is significantly higher than in healthy individuals.⁶² This increase in apoptosis immediately raises the possibility to target proapoptotic markers and activators such as caspase 3 using RNA knockdown techniques. When expressed in hippocampal cultures, ribozymes designed against pro-caspase 3 successfully protected against staurosporine-induced apoptosis.⁶³ Similarly, in an animal model for apoptosis; rat optic nerve transection, pretreatment of siRNAs targeting c-jun and apoptotic protease-activating factor-1 (Apaf-1) resulted in a significantly improved survival of retinal ganglion cells.⁶⁴ Blockade of apoptotic enzymes has been shown to be beneficial in other model systems as well. For example, siRNA designed against FAS-ligand expressed in a basal cell carcinoma, was shown to reduce the level of apoptosis of infiltrating immune cells and led to a more efficient clearing of the tumor cells.⁶⁵

Animal models of PD that completely recapitulate the human disease are not available. Toxins such as MPTP, 6-hydroxydopamine, and rotenone have been used to create an acute level of oxidative stress in the SN, causing DA neurons to die. Models such as these have mainly been used in cell-replacement/survival studies. Other models include -synuclein transgenic animals, which display a relatively wide array of pathology, but not resembling that of PD. In addition, overexpression of wt- synuclein or A53T using nigral-specific rAAV vectors induces over 50% nigral cell death and approximately an equal amount of striatal DA depletion in rats and monkeys.^{66, 67}

Parkin knockout mice have been created, but again they do not mimic the real disease. As a matter of fact, parkin-deficient animals display no significant pathology at all, which is surprising for a protein that is so ubiquitously expressed throughout the body. It is possible that utilizing siRNA to regulate certain genes may actually provide us with better models to study PD. For instance, in the case of parkin, this would allow one to study cellular impairment with spatio-temporal precision, and also allow one to avoid any 'genomic compensation' that may have occurred throughout the development of an organism.

Previous studies in our laboratory tried to test this hypothesis. We designed a number of ribozymes against parkin. Ribozymes are enzymatic RNAs that detect certain sequences in the mRNA and cleave them. These ribozymes were then packaged into rAAV and unilaterally injected into the SNc of adult rats. Immunohistochemistry, 4 weeks following the injections, clearly shows transduced cells (as detected by green fluorescent protein), with a significant loss of parkin expression (Figure 1a–g). However, attempts to identify accumulation of substrates and quantifying levels of parkin expression were unsuccessful. This was the result of an inherent difficulty with RNA and protein levels specifically in the SN. Proteins such as parkin are ubiquitously expressed in the brain, but the rAAV transduction pattern that we experience is highly specific for the SNc; thus it becomes very difficult to isolate these nigral neurons, and to analyze their content without an overwhelming background from surrounding tissue. Whereas studying other gene products such as -synuclein, which is highly upregulated in the SNc as compared to surrounding cells, is less difficult (Figure 1j–m).

Figure 1.

Ribozyme against parkin delivered via rAAV2 in substantia nigra with GFP coexpression This figure shows a representative animal that received unilateral nigral injections of rAAV-rbz131-IRES-GFP 4 weeks earlier (a–d). (a) Direct GFP fluorescence as a result of AAV transduction. (b) Tyrosine hydroxylase (TH) was labeled with Cy5 (shown in false color blue here). (c) Parkin is shown in red (Cy3). (d) Merger of a–c. In (a–d), yellow arrows point to GFP+ cells that are also TH+ but do not show parkin expression. White arrows show GFP+ cells that are also TH+ but do show parkin expression. In raw images, parkin expression in the injected hemisphere was barely detectable. On the uninjected side (e–g), all TH+ neurons had robust parkin expression. This figure shows that there is a variable amount of parkin knockdown in transduced neurons. We interpret this to mean that different nigral dopamine neurons received differing copy numbers of the ribozyme cDNA. This is a feature of the use of rAAV that cannot be controlled. Also, knockdown of parkin apparently caused no loss of TH positive neurons on the injected side. (h) In situ hybridization from an oligo-probe against rat parkin from another animal in this experiment, showing little parkin knockdown. (i) Similar in situ hybridization using a different oligo-probe in the same animal showing almost complete knockdown. We interpret these data to demonstrate that different parkin isoforms were differentially knocked down, and this should be considered for each gene in future siRNA experiments. Scale bar in (c) is 50 m and applies to a–g. Scale bar in (h) is 1 mm and applies to (i). (j–m) distribution of -synuclein (j, k) and parkin (l, m) in normal rat SN. (j) Low-power photomicrograph of -synuclein staining in normal rat SN. There is relative overexpression of -synuclein in the substantia nigra. The white triangle shows the area of enlargement shown in (k) and also shows that there is intense fiber staining in the striatonigral fibers. Scale bar=1 mm and applies to (l). (k) Magnification of the nigral neurons expressing -synuclein. The white triangle is pointing to striatonigral fibers that also have relative overexpression of -synuclein. Scale bar=100 m and applies to (m). (l) Low-power photomicrograh of parkin staining demonstrating more ubiquitous and homogenous parkin staining in the SN. The white triangle shows the area of enlargement shown in (m). (m) Magnification of parkin staining. Note that some cells resembling microglia show some specific staining. (j–m) are intended to illustrate the problem when using siRNA strategies for specific cell populations like nigral DA neurons, Western blots or RT-PCR for -synuclein will be relatively informative after taking punches from SN, while these same methods will be too diluted by other cell phenotypes when dealing with parkin knockdown.

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RNA interference has been and will continue to play an important role in PD research. However, the ultimate goal of researchers is to bring treatments and cures to the clinics. Given the relative ease by which one can design a siRNA to target a certain gene, there is no need for lengthy pharmacological analyses, and drug targeting issues. One can simply target the specific circuits in the brain using viral vectors expressing an siRNA. The potential targets in PD are numerous. First of all, one could target -synuclein, which seems to be intimately tied to the disease. The substrates of parkin are also attractive targets. Blocking the production of various apoptotic activators also become potential treatment strategies, but this approach also raises some concern about the risk for cancer if the siRNA is expressed in glia.

For example, can a delivery system be refined enough, to target only dopaminergic cells? What happens if an antiapoptotic siRNA is expressed in the surrounding glia? What if there is a gain of function mutation of an otherwise required protein? Lessons learned from ribozyme studies demonstrate that one can target the mutated sequence specifically, but if there is extensive allelic heterogeneity among disease mutations, this approach becomes less feasible. In the event that the mutant allele cannot be targeted, one can target a common wild-type sequence, and attempt to replace the gene with a siRNA or ribozyme-resistant copy. Another concern is the efficacy with which you can knockdown a certain mRNA. Even in the most favorable circumstances 100% reduction is not feasible. Thus, experiments must be designed to determine if residual expression of the gene is still therapeutic.

For instance, one of the earliest successful in vivo siRNA experiments targeted tyrosine hydroxylase (TH) expression in the SNc of mice using rAAV-mediated expression. TH is the rate limiting enzyme in the production of DA, the main signaling molecule in this circuit. Although, the experiment showed a significant reduction in TH levels, the behavioral testing was not entirely consistent with complete DA depletion in the nigrostriatal tract.⁶⁸ These results emphasize the point that in some instances siRNA may not be enough to achieve the intended biochemical result. An additional concern comes with that of 'off-targeting'. Although great care is practiced when designing the siRNA, you may still be affecting other genes, effects that may or may not have detrimental results.

Conclusion

Although in its infancy, applied siRNA technology has had great success in the laboratory, both to study pathology and to effect a cure. However, as of yet, only one clinical trial is underway, targeting vascular Endothelial Growth Factor Receptor-1 in age-related macular degeneration. In PD, there are numerous potential targets for RNA knockdown that when reduced could slow or even halt the progression of the disease (Figure 2).

Given the recent high-profile successes of virally delivered siRNA, this strategy is clearly poised to burst onto the PD research scene. All of the PD strategies to impact survival of DA neurons require nigral DA neuronal expression of the siRNA. Currently, the rAAV2 vector system has shown the most specificity for nigral DA neurons. In this review, we have pointed out some caveats when dealing with ubiquitously expressed proteins in the nigra (Figure 1). These potential problems in demonstrating knockdown must be taken into account in future studies.

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Acknowledgements

This work was supported by a Fast-track grant from the Michael J Fox Foundation to RJM and ASL.