Roles of microRNAs in Parkinson’s and other neurodegenerative diseases

CHAPTER

Roles of microRNAs in Parkinson’s and other neurodegenerative diseases

8

Qingxuan Lai⁎, Nicola Murgia⁎, Ilmari Parkkinen†, Andrii Domanskyi†, Ilya A. Vinnikov⁎ Laboratory of Molecular Neurobiology, Sheng Yushou Center of Cell Biology and Immunology, Department of Genetics and Developmental Biology, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, China⁎ Institute of Biotechnology, HiLIFE, University of Helsinki, Helsinki, Finland†

1 ­ Introduction Multiple factors contribute to the onset and progression of neurodegenerative (ND) diseases including genetic mutations, posttranscriptional dysregulation, and environmental influence [1, 2]. MicroRNAs are small noncoding RNAs adjusting expression of genes participating in the development and physiology of many tissues including central and peripheral nervous systems. Due to the unique characteristics of neurons including local translation and RNA interference (RNAi) in the neurites, microRNAs play a crucial role in neuronal physiology and pathophysiology [3]. Here, we highlight the age-related and disease-specific expression changes of neuronal microRNAs and their possible roles in Parkinson’s disease (PD) and other ND diseases.

2 ­ ND Diseases and Their Etiology Progressive death of neuronal populations in the brain and/or spinal cord is the main characteristic of ND diseases [4]. These include Alzheimer’s disease (AD), PD, Huntington’s disease (HD), dementia with Lewy bodies (DLB), and other disorders impacting tens of millions of persons worldwide [5]. The onset and progression of some of these diseases critically depend on genetic factors, such as an expansion of cytosine-adenine-guanine (CAG) triplets in specific genes leading to neurodegeneration via aberrant expression of so-called polyglutamine, or polyQ proteins. The most prominent of such polyQ pathologies is HD with polyQ repeats in the huntingtin protein [6, 7]. Another big group of ND disorders comprises prion and prion-like diseases in which the aggregation of prion-like proteins such as amyloid β [8], tau-protein [9] or α-synuclein [10] damages neuronal function and spreads to adjacent healthy neurons. Structural, biophysical, and biochemical characteristics AGO-Driven Non-Coding RNAs. https://doi.org/10.1016/B978-0-12-815669-8.00008-7 © 2019 Elsevier Inc. All rights reserved.

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of the above-mentioned proteins are very similar to prions which themselves cause transmissible spongiform encephalopathies [11–13]. Indeed, accumulation of amyloid β and tau is one of the key pathological features of AD [14], the most frequent ND disorder leading to progressive devastating dementia and death [15]. Similarly, aberrant accumulation of α-synuclein aggregates [10] and formation of Lewy bodies may lead to the degeneration of dopamine (DA) neurons and PD, the second most common ND disorder.

3 ­Epidemiology of PD PD is the most common movement disorder manifested by locomotor and posture disturbances and later also mental deterioration, as well as a wide range of nonmotor symptoms including sleep disturbance, constipation, and anosmia [16, 17]. This disorder, common in all geographical locations and races (prevalence in the general population 0.3%), is known since 1817 as the “Shaking Palsy” when it was described by James Parkinson and further studied by Jean-Martin Charcot. PD’s prevalence ranges between 1% and 2% of the population above 60–65 years [18, 19]. This ND is ~1.5 times more frequent in men than women [20], with the age at onset ~2 years later in women than in men [21]. Aging population and increased life expectancy in the developed countries are leading to the increase in incidence of PD. It is estimated that about 10 million people are suffering from PD worldwide, and the annual incremental costs of PD treatment in the US approach $22,800 per patient [22]. Economic modeling shows that even modest reduction in PD progression rate could produce significant monetary benefits [23]. As prevalence of PD is increasing and population is aging, the PD treatment market size is expected to grow and may reach $5.69 billion by 2022.

4 ­Factors Contributing to Onset and Progression of PD Major motor symptoms of PD, such as tremors, bradykinesia, muscle rigidity, and postural instability, are caused by progressive age-related degeneration of DA neurons in the substantia nigra pars compacta (SNpc) [24, 25]. These cells together with ventral tegmental area DA neurons form neuronal networks regulating complex behaviors, emotions, and voluntary motion [26]. Enhanced aggregation of prion-like proteins associated with several ND pathologies including PD can be a consequence of genetic alteration, epigenetically caused overexpression or alternative splicing of the gene product, as well as aberrant folding, cleavage, or secretion [27, 28]. Hence, many ND diseases have a multifactorial etiology with contributions from genetic, epigenetic, and environmental factors [29–33]. Familial, somatic, and mitochondrial mutations, aging, exposure to toxins or specific diets, trauma, gender, accumulation of reactive oxygen species, aberrant gene products and toxic metabolites, and misfolded proteins, as well as dysfunction

5 ­Therapeutic strategies for Parkinson's disease

in mitochondrial, proteasome, autophagy functions, and alteration of glial support, immune responses, and vasculature function—all these factors may contribute to neurodegeneration [33–39]. Due to specific electrophysiological properties of ion channels and their pacemaking activity [40], toxicity of DA metabolites [41], and production of high levels of reactive oxygen species in the mitochondria [42], DA neurons in the SNpc are especially vulnerable to stress caused by aging, mitochondrial mutations, oxidative stress, accumulation of toxic metabolites, and other factors [43]. The majority of PD cases are sporadic with only about 10%–15% inherited. Genome-wide association studies (GWAS) have identified a number of the PDassociated autosomal recessive and dominant mutations in several proteins including α-synuclein, where several mutations can promote the formation of phosphorylated oligomers and insoluble neuronal aggregates [44]. The presence of Lewy bodies, intraneuronal inclusions containing aggregated and phosphorylated α-synuclein and ubiquitin as main components, is a characteristic feature in the brains of both sporadic and familial PD patients [44, 45]. Accumulation of phospho-α-synucleinpositive neuronal aggregates can compromise neuronal functions already at the early stage of PD by causing synaptic and mitochondrial dysfunction, cell stress, and dysregulation of protein degradation pathways [45]. Furthermore, Lewy bodies spread in the brain over time [46] as demonstrated by accumulation of Lewy bodies in neurons transplanted into the brains of PD patients, putatively, through transmission of misfolded prion-like proteins present in PD patient’s brain [47, 48].

5 ­Therapeutic Strategies for Parkinson's Disease Therapeutic approaches to treat PD include administration of ­ l-DOPA +  Carbidopa + catechol-O-methyltransferase (COMT) inhibitors and/or DA agonists at early and middle stages and surgical interventions such as deep brain stimulation and neural transplantation for advanced disease stages. At present, the treatment of PD by L-DOPA [49], deep brain stimulation [50], and other methods [51–53] can only alleviate the symptoms. The principle of L-DOPA treatment is to provide sufficient amounts of this DA precursor for the surviving DA neurons, and thus, to compensate for the lost ones. L-DOPA medication is associated with a variety of negative side effects and it usually loses effectivity 5–10 years after the first administration [54]. In this case, the deep brain stimulation therapy can alleviate the motor symptoms, but neither therapy can restrain nor reverse the progression of neuronal loss [55]. With the recent progress in the development of protocols for differentiation of functional dopaminergic neurons from human embryonic or induced pluripotent stem (iPS) cells [56, 57], transplantation of DA progenitor cells to PD patients will soon be tested in clinical trials [58]. However, only a fraction of transplanted cells survive [59] and integrate into existing neuronal circuits. There are promising reports about cell transplantation techniques which have not yet been approved for clinical

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use in PD [60–62]. However, despite established approaches of iPS cells differentiation into DA neurons and efficient survival of engrafted cells in the tissue [50, 61], more basic and preclinical research is required to increase the effectivity of treatment and address the risks. Due to the increase of the aged population around the world, there is an urgent demand for novel therapeutic approaches to treat PD and other ND pathologies. These approaches might come from a deep mechanistic insight into the onset and progression of these diseases. MicroRNAs are one of many factors possibly affecting the course of neurodegeneration and deciphering their roles in the pathology progression would be beneficial for basic and clinical research as well as the patients and the society. Although still in early stages of development, many microRNAs and short interfering RNAs (siRNAs) are being studied as potential therapeutics for the central nervous system disorders [63, 64].

6 ­RNAi Pathways: Mechanisms, Physiology, and Application for Therapeutic Gene Silencing The mammalian transcriptome is regulated by long noncoding RNAs such as lincRNAs and circular RNAs, and small noncoding RNAs such as microRNAs and small nucleolar RNAs (snoRNAs), e.g., small Cajal body-specific RNAs [65]. Notably, type III RNAse Dicer can employ certain snoRNAs as substrates, for example the h­ uman microRNA-like snoRNA ACA45 [66], while other snoRNAs have been implicated in a ND disease, Angelman syndrome [67]. The landscape of noncoding RNAs is vast; therefore, this chapter will only cover those which are processed or associated with Dicer and recruited by argonaute (Ago) proteins [68–72], namely microRNAs.

6.1 ­MicroRNAs and RNAi The first microRNAs had been discovered in the early 1990s, but their function remained unclear for many years [73, 74]. In the subsequent years, RNAi was extensively studied [75–78] and this evolutionarily conserved mechanism has been found to be very important in the development and homeostasis of plants and animals, regulating many cellular functions [65, 79, 80]. In mammals, RNAi is involved in various cellular processes including silencing genes and transposons, regulating heterochromatin formation, and maintaining genomic integrity through endogenous siRNA, microRNA, and piRNA pathways [65, 69, 80, 81]. MicroRNAs are abundant in mammals with over 5000 detected in the human genome, of which at least 50% are specific to humans [82]. A single microRNA can regulate hundreds of genes, and conversely, a single gene can be regulated by hundreds of microRNAs [83, 84]. This is partly due to microRNAs being, in most cases, only partially complementary to mRNAs, and therefore, effects of most microRNAs are subtle and do not completely abrogate the expression of a gene, but rather fine-tune its expression [84, 85]. Expression can thus be further downregulated

6 ­ RNAi pathways

by other cooperating microRNAs. Moreover, microRNAs may additionally regulate each other or even genes crucially involved in microRNA biogenesis, making the fine-tuning process even more sophisticated [86, 86a, 117]. Interestingly, highthroughput screening of microRNAs revealed that many of them are not functional unless they are very abundant adding to the complexity of their nature as regulators of the transcriptome and proteome [87]. MicroRNAs are predicted to affect the expression of more than half of all the protein-coding genes in the human genome [88]. Thus, one overarching function of microRNAs seems to be that they control protein expression noise [89]. Another, seemingly contradictory property of microRNAs is that they may upregulate translation, at least during cell cycle arrest [90]. This may be mediated by binding to the ­5′-UTRs and coding region of transcripts possibly regulating splicing and thus adding a yet another layer of regulatory complexity [91, 92].

6.2 ­PIWI Interacting RNAs PiRNAs are 24–32 nt long endogenous noncoding RNAs [94, 95]. The etymology behind piRNAs derives from the finding that they interact with the PIWI (P-elementinduced wimpy testis) subfamily of Ago proteins. They are best-known for regulating transposable elements in germline cells. More specifically, piRNAs target transposons and cleave them in complexes with PIWI-proteins. This may generate additional piRNAs in turn cleaving further transposons thus preventing detrimental modifications to the genome. Additionally, they are important regulators of chromatin dynamics. Recently, various biological roles of piRNAs have been elucidated in somatic cells, for example, in the context of memory formation and cancer [94, 96].

6.3 ­Artificial Substrates for RNAi and Their Application in Research and Therapy RNAi functions also with classes of artificially synthesized RNA mimicking intermediate molecules in the RNAi pathway biogenesis [81]. In addition to chemically stabilized microRNA mimics which could be injected or continuously infused into the brain [97], these include single- and double-stranded siRNAs and short hairpin RNAs (shRNAs). Application of 20–25 nt siRNAs for gene silencing in mammalian cells was demonstrated around two decades ago [75]. In contrast to microRNAs, siRNA binding leads predominantly to degradation of its target mRNA due to the full complementarity of siRNA/mRNA sequences [98]. Artificial siRNAs are widely used in biotechnological or medical applications [63, 99]. SiRNAs can be designed using various web-based tools optimizing them for specificity and efficiency. Apart from being used as single-stranded siRNAs, they can also be expressed within a pri-/pre-microRNA or shRNA backbone. Although artificial shRNAs are usually expressed by pol III promoters, such as the U6 promoter, they can also be designed to be transcribed by pol II, similar to pri-microRNAs [100]. SiRNA-mediated high-throughput screening approaches represent a useful tool to

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identify the functions of genes and signaling pathways [101]. One of siRNA-based therapeutics, patisiran, targeting transthyretin (TTR) to treat hereditary ATTR amyloidosis has been approved by FDA to become the first siRNA-based therapeutic to reach the market [102].

7 ­Neuronal MicroRNA Pathway in Physiology and Pathology The integrity of microRNA pathways is crucial for the maintenance of normal physiological function in the central nervous system. Fine-tuning of gene expression by microRNAs contributes to the orchestration of neuronal functions and cellular pathways [103, 104]. MicroRNAs regulate posttranscriptional expression of target proteins that are involved in neurite outgrowth and synapse formation. Both on patient-derived materials as well as in several animal models of PD, AD, and ALS, it was shown that dysregulation of microRNAs can lead to neurodegeneration [105, 106]. Even upon targeting single genes, microRNAs can strongly impact physiology of the tissue or disease progression. Indeed, suppression of actin regulator Lim-domain containing protein kinase 1 (Limk1) by miR-134 is sufficient to restrict the size of dendritic spines [107]. Likewise, miR-132 inhibits GTPase activating protein p250GAP which is necessary for neuritogenesis, formation of synaptic connections, and synaptic plasticity [108, 109]. In another study, overexpression of miR-185 in an in vitro model of PD showed suppression of autophagy and apoptosis [110]. Recently, it has also been discovered that there are plenty of microRNAs specifically expressed in the brain and crucial for their function by regulating local translation in the synapses, for example, by stabilizing a transcript and thus increasing its half-life [93].

8 ­Dicer and Its Role in Aging and Neurodegeneration Dicer, the ribonuclease crucial for microRNA biogenesis, is encoded in humans by the DICER1 gene. It is a multi-domain RNA-binding protein [111] belonging to the family of type III RNAse enzymes. Dicer dysfunctions and abnormal microRNA processing have been linked to aging and various ND diseases [112–116]. In particular, Dicer levels are downregulated in patients suffering from age-related diseases, such as PD and ALS. Expression of Dicer is tightly regulated, for example, by let-7a microRNA, thus representing a negative feedback loop [117]. In general, factors affecting the lin28/let-7 axis can rapidly alter Dicer and subsequently lin28 expression levels in brain-derived neurotrophic factor (BDNF)-dependent manner [118]. As Dicer is crucial in many cellular processes, it is not surprising that its deficiencies have been associated with many diseases [111, 119, 120]. DICER1 deficit can cause retinal cell degeneration in an advanced form of age-­ related macular degeneration in humans. Accordingly, conditional ablation of

9 ­ Dicer as a target to treat neurodegeneration

Dicer, but not seven other microRNA-processing enzymes, leads to degeneration of the same cells in mice [121]. Dicer dysfunction or downregulation is also apparent in many other CNS-related disorders. Decreased DICER1 in the blood has been detected in patients suffering from psychiatric conditions, e.g., posttraumatic stress disorder and depression [122]. Dicer is additionally downregulated in aged tissue, such as adipocytes [123] and globally in the brain [123]. Specific ND conditions have been linked to Dicer deficiencies. For example, Dicer ablation in oligodendrocytes promotes neuronal degeneration in mice, while low levels of Dicer in the blood are associated with multiple sclerosis [115, 124]. Moreover, microRNAs expression is downregulated in motor neurons of ALS patients, while Dicer stimulation treatment delays onset of symptoms in mouse ALS models [114]. Additionally, mRNA profiling on postmortem laser-capture microdissected (LCM) DA neurons of PD patients revealed downregulation of DICER1 compared to healthy individuals [125]. Furthermore, at least in two separate studies, conditional Dicer knockout mice have been shown to cause severe nigrostriatal dopaminergic cell loss (Fig.  8.1), demonstrating the importance of Dicer for DA neuron survival [112, 126]. Interestingly, in the same study, we identified that 87-week old mice have a significant reduction in Dicer1 mRNA in the ventral midbrain compared to 6.5-week old mice, which was also functionally linked with the predominant depletion of microRNAs in DA neurons. Dicer also seems to be critically involved in stress responses and is in turn itself vulnerable to stress conditions. Factors such as reactive oxygen species, hypoxia, and UV light may downregulate Dicer (Fig. 8.2) [127]. In contrast, Dicer protein levels are elevated in response to mild hyperthermia [128]. β-catenin, which is implicated in stress-related psychiatric disorders, has been shown to interact with Dicer and mediate proresilient effects [129].

9 ­ Dicer as a Target to Treat Neurodegeneration There are many factors and compounds shown to affect RNAi and a few known compounds modulating Dicer activity (Fig. 8.2). Dicer is upregulated by a common diabetes drug, metformin, and is required for metformin’s action in cellular senescence models [130]. Metformin is also protective in several animal models of PD; however, since metformin is a polypharmacological compound, it remains to be shown whether this effect is mediated by its ability to upregulate Dicer [131, 132]. Thapsigargin, a useful pharmacological research compound, is a known regulator of endoplasmic reticulum (ER) stress produced by the plant Thapsia garganica [133]. It raises cytosolic calcium and also was shown to inhibit Dicer [114]. One of the best studied small-molecule compounds activating Dicer is a fluoroquinolone antibiotic enoxacin. It is a bacterial DNA gyrase and topoisomerase IV inhibitor, which was one of the main reasons to be chosen for studying its RNAi enhancing abilities, since inhibiting RNA helicases may stabilize dsRNAs and thus

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LoxP LoxP Dicer1

5'

3'

Dopamine neurons

+

Pre-microRNA

X

X

Dicer1 ice

Dicer Dic

tre

mo r

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DatCreERT2

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Parkinsonian phenotype

Neurodegeneration in the substantia nigra

FIG. 8.1 Inducible Cre-LoxP genetic model for selective deletion of Dicer in adult dopamine neurons implemented in the study by Chmielarz et al. [112]. Cre recombinase fusion with mutated estrogen receptor ligand binding domain (CreERT2) makes it tamoxifen-inducible. After binding to tamoxifen, ERT2 domain undergoes conformational change and dissociates from chaperone proteins, allowing CreERT2 to enter the nucleus and recombine LoxP sites flanking the target gene (in this case Dicer1). CreERT2 is expressed in dopaminergic neurons under control of dopamine transporter (Dat) gene promoter in bacterial artificial chromosome (BAC) transgenic construct, allowing highly specific transgene expression. By injecting tamoxifen to adult (8–10 weeks old) mice, the researchers can induce Dicer deletion at specific time point, allowing to preserve Dicer function during embryonic and postnatal development. Deletion of Dicer in adult dopaminergic neurons causes their progressive degeneration 4–10 weeks after tamoxifen treatment.

e­ nhance RNAi [134]. Enoxacin activates Dicer indirectly by enhancing its ­interaction with transactivation response element RNA-binding protein (TRBP, TARBP2) [135]. Also, enoxacin has neuroprotective effects on midbrain DA neurons and protects against ER stress [112]. Additionally, it corrects ALS-associated microRNA dysregulation in vitro and is beneficial for neuromuscular function in mouse ALS models [114]. Furthermore, enoxacin is considered as a possible medication for ALS by the European Medicines Agency (Public summary of opinion on orphan designation EMA/COMP/125722/2015). Enoxacin also prevents learned helplessness in the rat, suggesting that the correction of microRNA dysregulation by activating Dicer could also be beneficial in the treatment of psychiatric disorders [136].

10 ­Effect of aging on microRNA pathway in dopamine neurons

FIG. 8.2 Aging and stress affect levels and activity of Dicer. Multiple physical and chemical stressors affect microRNA processing activity of Dicer complex by inducing phosphorylation of its component proteins and destabilizing their interaction with Dicer. The expression levels of Dicer, as well as multiple microRNAs, also decline with aging. Dysregulation of microRNA network affects cellular homeostasis, impairing stress response, and survival pathways. This creates a vicious cycle whereby increased stress leads to destabilization of microRNAs which, in turn, contribute to increased stress and eventual cell death. BDNF, brain derived neurotrophic factor; miRNAs, microRNAs; PACT, protein activator of interferon induced protein kinase EIF2AK2; ROS, reactive oxygen species; TRBP, trans-activation responsive RNA-binding protein; UV, ultraviolet radiation.

Enoxacin has many targets and activates Dicer at relatively high concentrations compared to its antibiotic activity, so it is prone to causing various side effects which limits its therapeutic potential as an RNAi enhancer. These side effects include seizures and defects in bone formation [137, 138]. Furthermore, due to its antibiotic activity, dysbiosis may per se contribute to neurodegeneration, as dysfunctional microbiome has been linked to PD [27]. Therefore, screening of compounds activating Dicer might help develop new approaches to treat ND diseases.

10 ­Effect of Aging on MicroRNA Pathway in DA Neurons of the Ventral Midbrain Several studies reported an age-related decline of microRNAs in both human and other vertebrates [139]. For example, miR-22, -101a, -720, -721, -30d, -34a, -468, -699b, and -709 were downregulated in the aged mouse brain [140]. As mentioned above, we revealed an age-related downregulation of midbrain Dicer1 in mice [112]. Interestingly, DICER1 gene is also downregulated in DA neurons from PD patients

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[125]. Aging is the major factor contributing to PD, so predominant age-related downregulation of microRNAs [105] in DA neurons [112] might be an important factor contributing to the progression of neurodegeneration [105]. Indeed, when recapitulating the physiological impact of microRNA downregulation by knocking out just one allele of Dicer1 in DA neurons, we observed a progressive decrease of striatal DA and its metabolites in comparison to control mice, with apparent differential vulnerability in male vs. female mice which was similar to a gender bias in PD. Moreover, inactivation of both alleles resulted in mitochondrial reactive oxygen species accumulation in DA neurons, progressive devastation of SNpc, and reactive astroglyosis [112, 126] accompanied by severe locomotor and postural deficits, tremor, and rigidity in analogy with typical PD symptoms: bradykinesia, rest tremor, rigidity, postural, and gait impairments [141, 142].

11 ­MicroRNAs Associated With DA Neuron Degeneration and PD Recent GWAS have largely contributed to identification of genetic factors associated with PD. Discovery of PD-related single nucleotide polymorphisms (SNPs) helps researchers to decipher the molecular mechanisms behind the onset and progression of both familial and nonfamilial types of this disease. Some microRNAs have been predicted and validated to target the 3′-UTR regions of neuropathology-associated mRNAs [143]. SNPs in these genes might disrupt the binding of a microRNA to its target causing a pathological accumulation of the protein, thus triggering neuropathology. Several studies have deciphered specific microRNA expression patterns associated with PD (Fig. 8.3). All of them implemented postmortem brain tissues from PD patients. It is suggested that PD starts from the intestine and gradually spreads to peripheral and central nervous systems to predominantly hit SNpc but also other basal ganglia [144] followed by cortex, amygdala, and cerebellum, which are usually less affected in this neuropathology.

11.1 ­MicroRNA Expression Patterns in the Basal Ganglia of PD Patients The first study to assess the expression of microRNAs in three PD patients and five healthy individuals (of which midbrain samples were available only from three individuals). In this work, Kim and colleagues identified miR-133b to be specifically expressed in the midbrain (compared to the cortex and cerebellum) (Fig. 8.3). Moreover, they found miR-133b to be deficient in PD patients [145]. In this work, the authors also postulate an existence of Pitx3↔miR-133b feedback loop as well as the essential function of Dicer for developing DA system which was studied on in vitro and in vivo models [145]. Moreover, their work inspired other groups to study the involvement of microRNAs in neurodegeneration [146].

11 ­ MicroRNAs in dopamine neuron degeneration and PD

FIG. 8.3 MicroRNA expression is altered in the brains of Parkinson’s disease patients. MicroRNA expression profiling studies demonstrated changes in the levels of particular microRNAs in different brain regions in postmortem samples from PD patients, as compared to age-matched healthy individuals. These results clearly demonstrate dysregulation of microRNA network in PD; however, the task to elucidate particular molecular pathways affected by these changes proved to be very difficult, predominantly because of limited ability of current experimental methods to identify microRNA targets. LCM, laser-capture microdissection.

However, Briggs et al., the authors of the latest most prominent study in this field, did not manage to detect downregulation of miR-133b in LCM DA neurons from PD patients [147]. The very means by which the samples were collected makes this study by Briggs and colleagues truly unique and provides neurobiologists and neurologists with unprecedented glimpse into microRNA biogenesis alteration in the cells mostly affected by PD—DA neurons. In this study involving 5 vs. 5 male, 3 vs. 3 female PD patients, and healthy individuals, the authors reported for the first time a gender-specific patterns of microRNA expression. Indeed, Briggs and colleagues found that miR-106a, -135a, 148a, -223, -26a, -28-5p, -335, and -92a were upregulated only in males, while let-7b miR-106a and -95 were upregulated

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only in females compared to healthy individuals. Overall, this work represents an example of scientifically highly relevant target chosen by the researchers: to study specifically the neurons mostly affected by a ND pathology. We very much hope that in the future we will have more systematic analyses of coding and noncoding transcriptomes from distinct neuronal populations also for other ND diseases, which will be highly relevant for both basic and clinical research. Indeed, the patterns of both mRNAs and microRNAs are so distinct in different neuronal and nonneuronal populations as well as within subpopulations of neurons of the same neurotransmitter class that sampling of a large amount of tissue comprising tens of different cell types may indeed become an issue for experiment conclusions [148, 149]. For example, the significant downregulation of a given microRNA in a crudely dissected tissue may reflect: (i) its downregulation in the degenerating neuron which might be an interesting target for drug development, but only in case this change has a causative nature for the disease; (ii) the specificity of its expression in the degenerating population so that the microRNA is simply downregulated in the tissue due to the loss of the neurons which exclusively express it; (iii) a compensatory downregulation of this microRNA in spared suffering neurons, spared healthy neurons, or even in supporting cells so that this downregulation could be not detrimental, but beneficial for survival of the spared neurons. Thus, both possibilities (ii) and (iii) could reflect the discrepancies between LCM data from Briggs et al. [147] and midbrain data from Kim et al. [145]. Altogether, these points nicely illustrate the complexity of modern research studying genetic and epigenetic interrelations in ND diseases. Interestingly, Briggs and colleagues also did not detect downregulation of miR34b/c in DA neurons from PD patients [147]. This microRNA has been previously found by Miñones-Moyano and colleagues to be decreased in several areas of the brain—prefrontal cortex, amygdala, SNpc, cerebellum—in 29 PD patients compared to 33 healthy individuals [150]. The discrepancies might again reflect the abovedescribed possibilities (ii) and (iii). However, we also need to point out here a profound difference in numbers of patients used in both studies. Another highly relevant assessment of microRNA expression was performed by Nair and Ge on striatal tissues from 12 PD patients treated with l-DOPA and/or dopaminergic agonists [151]. Although they have not used any tissue from the midbrain, striatal samples might provide an important view on the pathogenesis of PD. Indeed, in the healthy brain, striatum is vastly and predominantly innervated by DA neuronal projections. Also, it is well-established that neurodegeneration in PD impacts the projections at first, followed by progressive death of neuronal bodies [152– 154]. Not only these data might provide the unique information about local functions of microRNAs in the DA neuron projections, but they might also point to microRNAs specifically expressed in DA neurons—as degeneration of DA neuron axons might be reflected by downregulation of DA neuron-specific microRNAs (see the possibility (ii) above). These data might also help decipher microRNAs specifically altered in suffering or healthy DA neurons of PD patients. Using nCounter Human

11 ­ MicroRNAs in dopamine neuron degeneration and PD

v2 microRNA expression assay kit, Nair and Ge found 13 altered microRNAs with PD-related expression alteration (Fig. 8.3) [151]. Similarly, Cho and colleagues identified downregulation of miR-205 in the frontal cerebral cortex and striatum of 16 PD patients with dementia compared to 7 healthy individuals [155]. The experiment was performed by implementing TaqMan qPCR array capable of distinguishing the mature from precursor forms of microRNAs— this approach was also used by Briggs et al. in their study discussed earlier [147]. Indeed, this might be important to know whether the expression of a microRNA is affected on the level of transcription, splicing, and RNAi or on the level of maturation and stabilization. Importantly, in this study, the authors generated the first highly specific antibodies against LRRK2 [155].

11.2 ­MicroRNA Expression Assessment in Other Tissues From PD Patients In most of the studies listed in the following section, assessments of microRNA-­target interaction have been discussed. However, none of them was conclusive enough to prove both validated microRNA-target interaction and in vivo reciprocal interrelations in the tissue; hence, we would only mention here the descriptive data from these publications and refrain to discuss their mechanistic analyses. The two studies discussed below did not sample basal ganglia, the regions mostly affected during the course of PD; hence, their assumptions and interpretations should be taken with caution. Instead, both of them used cerebral cortex tissues. In one of them, the authors again implemented TaqMan qPCR profiling of 744 microRNAs to reveal the upregulation of 43 microRNAs in the cingulate cortex from 22 Caucasian PD patients compared to 10 healthy individuals [156]. In another work, Hoss and colleagues compared the levels of 125 microRNAs in 29 PD patients and 33 healthy individuals with or without dementia [157]. Interestingly, 21 microRNAs were found to be differentially expressed in PD and HD, while only two of these (miR-10b-5p, miR-320b) were changed in opposite directions in one disease compared with the other. Due to the location of the tissue, prefrontal cortex, which is particularly affected in HD, we find this study more relevant for this pathology. The comparative analysis of all these studies is not easy due to several reasons. First, they are conducted in different regions of the brain, some of which have very limited involvement in PD pathology. With the exception of Briggs et al. study [147] which implemented LCM neurons for profiling, all others used tissues comprising a big number of different cell types, which makes it difficult to accurately conclude what the changes in microRNA levels could be attributed to. Second, low numbers of patients used for the profiling might question the relevance of the obtained data due to interindividual variation strongly impacting results in such low study groups. In order to achieve a profound insight on functional roles of microRNAs in ND ­diseases, we need novel approaches such as single cell transcriptome analyses, HITS-CLIP

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analyses, improved in vivo imaging techniques, animal studies, and models involving DA neurons differentiated from patient-derived iPS cells.

12 ­Conclusions Despite years of research, the challenge of halting the progression and/or reversing the loss of neurons in ND diseases remains largely unsolved; hence, further studies of molecules, cellular pathways, and neuronal circuit interactions that can support functions and survival of neurons in aging and neurodegeneration are required. Posttranscriptional regulation of gene expression by microRNAs affects multiple cellular pathways regulating stress response, survival, and neuronal functions. MicroRNA biogenesis is impaired in stress and aging and, therefore, it is plausible to suggest that the loss of particular microRNAs may contribute to increased vulnerability of certain neuronal populations to age-related stressors. Indeed, several studies involving PD models have demonstrated the importance of microRNA biogenesis in general and individual microRNAs in particular for the maintenance, stress response, and survival of adult DA neurons. Selective deletions of individual genes crucial for microRNA biogenesis in these cells clearly showed that disturbances of microRNA regulatory network impair functions of these neurons leading to their progressive loss and PD-like phenotype. Expression profiling studies identified several individual microRNAs to be crucial for differentiation and survival of DA neurons. These studies essentially support the idea of using either individual microRNAs or their combinations and/or stimulating microRNA biogenesis as new therapeutic options to promote survival of DA neurons in PD. Despite these advances, many challenges remain on the road to successful microRNA-based PD therapy. Difficulties in predicting microRNA targets translate into problems with possible unspecific side effects for particular microRNA mimics. Specific targeting of DA neurons, stability of microRNA mimics [158], and their ability to cross the blood-brain barrier that would allow systemic delivery are important factors that need to be considered and optimized for a successful therapy approach. General stimulation of microRNA biogenesis may be an alternative option to achieve neuroprotection. Indeed, Dicer is a promising drug target for many microRNA dysregulationassociated diseases. For example, cancers and age-associated ND diseases, particularly ALS and PD, were shown to be linked to Dicer deficiencies [112, 114, 120]. Inducing (activating or upregulating) Dicer could help prevent global dysregulation of microRNA network and promote survival of neurons under stress and aging conditions. Therefore, compounds which selectively induce Dicer could have therapeutic value as was demonstrated, for example, by enoxacin protecting motor neurons in an ALS model and DA neurons in a PD model. However, better and more potent Dicer activating drugs with fewer side effects will be desirable as a more promising candidate for ND disease therapy. Thus, developing functional, sensitive, reliable, and scalable assays to measure the pre-microRNA-processing activity of Dicer and screen for Dicer stimulating compounds would be very helpful. Moreover, further studies

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refining the mechanisms underlying interactions of microRNAs with their targets will help identify the best candidate neuroprotective microRNAs with fewer unspecific effects. Based on the current advances in PD research, we envision the development of combinatorial therapies which could consist of small molecules and neurotrophic factors stimulating essential prosurvival pathways and/or microRNA biogenesis, as well as individual neuroprotective microRNAs, to rescue and support DA neuron functions and survival in PD and/or other ND disorders.

­Funding This work was funded by Shanghai Jiao Tong University grant #AF0800059 to Q. L.; Academy of Finland grants #293392 and #287843, and HiPOC grant #797012303 to A. D.; National Natural Science Foundation of China grant #31771433, Joint grant from Sheng Yushou Center of Cell Biology and Immunology donated by Ms. Sheng Chenghui, and Shanghai Jiao Tong University grant # AF0800056 to I. A. V.

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