**4. Conclusions**

306 Etiology and Pathophysiology of Parkinson's Disease

the substantia nigra and promotes the survival of dopaminergic neurons. A subsequent Comparative Genomic Analysis Study (CGAS) reported in 2004 identified rs1989754 within FGF20 as associated with PD (van der Walt, 2004). Therefore, Wang et al. (Wang, 2008) selected FGF20 for further association testing using a family-based design. The authors observed that the strongest association (P = 0.0001) occured with rs12720208 [C/T], a SNP that was demonstrated as mediating the allele-specific *in vitro* targeting of miR-433 to the 3'UTR of *FGF20*. The authors also showed that miR-433 is highly expressed in the brain. Unlike prior studies, these researches went even further by providing human *in vivo* validation and testing a molecular mechanism by which differential miR-433 targeting potentially leads to PD. In cell culture experiments and in PD brains, increased translation of

Two independent GWASs for PD did not test rs12720208 (Fung, 2006, Maraganore, 2005). However, one of these studies tested a nearby SNP (rs1989754) and found no signal for association (P = 0.40) (Fung, 2006). The other study also tested another SNP within FGF20 (rs17515020) and observed a modest signal for association with PD (P = 0.03) (Maraganore, 2005). However, this result was not significant after correction for multiple testing (Maraganore, 2005). According to the phased HapMap CEU genotype data, rs12720208 is not in strong LD with either rs1989754 (r2 = 0.1) or rs17515020 (r2 = 0.029); therefore, this SNP may still be associated with PD independently. Given that this study was not confounded by population stratification because of the use of a family-based association study design, and given that there is compelling functional evidence for both allele-specific targeting and an underlying molecular mechanism for the role of miR-433–FGF20 in PD pathogenesis, rs12720208 is the most attractive human poly-miRTS for use in future replication studies (Sethupathy & Collins, 2008). However, there was no association between the *FGF20* SNP rs12720208 and Parkinson's disease in Spanish patients (de Mena, 2010). Overexpression of SNCA appears to be a common feature in PD and other αsynucleinopathies. In addition, it was demonstrated recently that miR-7 interacts *in vitro* with the 3'UTR of *SNCA*, represses the expression α-synuclein and inhibits α-synucleinmediated cell death (Junn, 2009). Of note, miR-7 inhibits the cellular susceptibility of neuroblastoma cells to oxidative stress induced by a mutant form of SNCA, providing evidence that miRNAs protect neuronal cells against cellular stress. The presence of miR-7 in the substantia nigra was also validated, thus supporting a physiological role for this molecule in DA neurons. However, miR-7 was initially reported as highly expressed in the mouse pituitary gland (Bak, 2008, as cited in Lau & de Strooper, 2010) and its expression was ~15-fold lower in the substantia nigra compared with the pituitary gland, leaving open the possibility that other miRNAs regulate dopaminergic neurons in amore dynamic and

One of the latest studies on this subject revealed a previously unknown mechanism of regulation of SNCA levels in the nervous system (Doxakis, 2010). Specifically, two brainenriched miRNAs, miR-7 and miR-153, were shown to bind directly to the 3'UTR of the *SNCA* mRNA and reduce its levels significantly. RNA and protein expression analysis showed developmental and tissue coexpression among miR-7, miR-153, and SNCA. The results of the study indicate that miR-7 and miR-153 have synergistic effect, require the 3'UTR of *SNCA* mRNA to regulate SNCA protein expression, do not interact with the coding region of *SNCA* to regulate the expression of SNCA protein, and act at the pretranslational level. Taking into consideration the two types of miRNA function, these findings strongly suggest that miR-7 and miR-153 play a role in the modulation/buffering of

FGF20 was correlated with increased expression of α-synuclein.

efficient manner (Lau & de Strooper, 2010).

SNCA protein levels in the nervous system.

Most neurochemical and gene expression studies on the mechanism of DA neuron death in PD and its animal models, have been conducted at a time when the majority of dopamine neurons are dead. However, the detailed profile of the crucial initial neurochemical and gene expression changes in injured (but not dead) neurons is more important, as the early genetic and biochemical alterations differ from those that occur at the time of neuronal death.

It is also important to emphasize that mRNA data reveal information on the transcriptional activation of genes, but do not provide much information on the actual protein levels and function. In addition, array data cannot predict whether deregulated gene expression is a primary or a secondary effect of cell function. For example, a gene could be down- or upregulated by factors such as miRNAs or transcriptional activators (or inhibitors), independent of its protein function and/or as a consequence of positive and negative feedback loops. Moreover, protein function relies on the interaction of down- and upstream factors within a pathway, i.e. downstream factors are more dependent on upstream signaling compared with upstream factors, which may influence a cascade of downstream events that can include multiple pathways. Thus, the consequences of deregulated gene expression are exerted on multiple levels within a complex and dynamic interplay of factors and mechanisms. Laser-microscopy-based microarray studies can only provide a "snapshot" of these events. Nevertheless, several studies showed that many genes associated with the pathogenesis of Parkinson's disease are deregulated in single captured postmortem DA neurons. This could provide a "molecular fingerprint identity" of a latestage DA neuron affected by sporadic Parkinson's disease. The striking downregulation of PARK genes is a key aspect. As their mutation-induced malfunction in the familial forms of Parkinson's disease rapidly accelerates DA neuron degeneration, the results from the studies reviewed may support the view that these genes are also involved in the pathogenesis of sporadic Parkinson's disease. Data also point to an imbalance in neuronal homeostasis and stress characterized by factors related to high metabolic rate,

Analysis of Transcriptome Alterations in Parkinson's Disease 309

This work has been supported in part by grants of the Russian Basic Research Foundation (Grants 07-04-01511, 07-04-00027, 09-04-01237-a), State Contracts (02.740.11.0084, P419, P1055), Russian Academy of Sciences program "Molecular and Cellular Biology" Russian

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neurotransmission and ion-channel activity. This stress may be part of the normal homeostasis and aging of DA neurons, but could be exacerbated in the presence of an unfavorable imbalance. In addition, the array data suggest a disintegration of key cellular functions, such as mitochondria-associated energy metabolism, protein degradation, synaptic function, and cytoskeletal integrity, revealing a cellular state that is characterized by programmed cell death. However, despite this cellular demise, some genes linked to survival mechanisms were upregulated, indicating the activation of compensatory mechanisms. Finally, the lack or the relatively modest deregulation of genes important for the DA neuronal phenotype suggests that the DA neurotransmitter identity (including DA production) seems to be sustained even when the neurons are severely damaged. It appears that the gene expression profile of the DA neurons of late-stage Parkinson's disease is consistent with the view that Parkinson's disease is a complex disorder and that multiple factors and cellular pathways are involved in its pathogenesis.

To date, whether miRNAs are also actively involved in the etiology or progression of neurodegenerative disorders remains uncertain. These small regulators clearly seem to be required for the survival of specific types of mature neurons in some model organisms; however, whether the loss of individual miRNAs can account for the drastic disease phenotypes remains to be determined. As many different cellular processes have been implicated in neurodegenerative disorders, the miRNAs involved in these pathways will obviously be found as misregulated in disease tissues. However, the degree to which their misregulation is causative in the diseases remains a pressing, but unanswered, question. Identification of causal links opens prospects for therapeutic intervention, perhaps by replacing missing miRNAs or blocking the activity of miRNAs that are overexpressed. Similarly, some miRNAs seem to have a neuroprotective role; therefore, they could potentially be used to prevent, or at least decelerate, the progressive loss of neurons in the diseased brain. These are tantalizing prospects that remain far from our grasp (Bushati & Cohen, 2008). Studies of miRNA in neurodegenerative diseases are emerging. Currently, both changes in the expression profiles of several miRNA and polymorphisms affecting the interactions between miRNAs and their targets are being addressed in various studies on neurodegenerative disease. It is difficult to determine if the changes in miRNA expression detected in the brains or cerebrospinal fluid (CSF) of patients are primary or secondary events, or both. Nevertheless early or late in the evolution of the disease, they may contribute to the pathogenesis of the observed lesions and neuronal loss. Unique patterns of miRNA expression in the CSF of particular neurodegenerative diseases may be useful as molecular biomarkers for disease diagnosis and, eventually, for the prediction of therapeutic responses. The identification of miRNAs that cause a specific pathology could open new therapeutic perspectives to block endogenous miRNAs or deliver exogenous miRNAs. To date either antisense oligonucleotides that are chemically modified (Meister, 2004) or expressed sequences corresponding to multiple miRNA seed targets (miRNA sponge) (Ebert, 2007) have been used as microRNA inhibitors. Delivery of these molecules to the central nervous system, while avoiding toxicities, may be the challenge of future research in this area. Furthermore, as specific nuclear or cytoplasmic protein accumulation causes the neuropathological manifestation in several neurodegenerative disorders, the identification of microRNAs that regulate the translation of these targets may represent the first step toward therapeutic applications. The second step might be the evaluation of the quantitative effects of specific amounts of "therapeutic" microRNAs on the proteome (Barbato, 2009).
