In the Drosophila brain, piRNAs, which were initially identified as piRNA-like molecules, were shown to be derived from TE sequences (31). Subsequently, piRNA-like molecules were also detected in the cerebral cortex of rhesus macaque (32). Furthermore, MIWI/piLRNAs and a type of piRNA were identified in the dendritic compartment of cultured mouse hippocampal neurons (33). Interestingly, the same study revealed that piRNAs regulate spine morphogenesis by targeting the coding regions of several neuronal mRNAs associated with spine formation. The Piwi/piRNA complex enhances memory-related synaptic plasticity by facilitating serotonin-dependent methylation of the CREB2 promoter in the Aplysia central nervous system (CNS) (34). Thus, piRNAs affect neurogenesis and synaptic plasticity by regulating gene expression at the transcript and genomic levels.

Long interspersed nuclear elements (LINEs) are transcribed at high levels in various rat tissues, including the brain, and their expression is controlled by DNA methylation and non-coding RNAs, including piRNAs (35). Therefore, piRNAs are suggested to have a role in the tissue-specific variation of genomes through the expression and mobilization of LINEs. The expression of TEs and PIWI proteins, such as Aub and Ago3, shows an inverse correlation in the αβ neurons of the mushroom body in Drosophila (11). Based on these findings, piRNAs regulate genomic heterogeneity in neurons by controlling TE transposition.

Defects in the PIWI protein MILI induce hypomethylation of LINE1 promoter regions and induce behavioral deficits in mice, such as hyperactivity and reduced anxiety (36). These effects are likely caused by the inactivation of piRNA biogenesis, although the precise roles of piRNAs in the CNS remain to be determined.

Altered expression of 105 piRNAs was identified in the cerebral cortex of ischemic rats compared with the sham group (37). Those piRNAs suppress the activation of TEs inserted in a variety of genomic regions. The piRNA promoter regions contain putative binding sites for several transcription factors, suggesting that piRNAs are regulated by transcription factors. The changes in piRNA expression observed upon focal ischemia might influence the activity of TEs and subsequent genomic changes, although researchers have not yet presented evidence that this event contributes to brain damage.

The biogenesis of piRNAs is activated during axon regeneration after injury (38.39). Rett Syndrome is a severe neurodevelopmental disorder, accompanied with intellectual disability in girls. Patients show developmental delay, including hand movements, language and communication. The expression of piRNAs is also increased by the loss of methyl-CpG binding protein 2 (MeCP2), mutations of which are responsible for Rett syndrome, in the murine cerebellum (40). Because piRNAs are expressed from TEs, TE activity and mobility might be increased in this model. Although the analysis was performed without replicated samples and further studies are warranted to confirm the findings, piRNAs represent potential clinical biomarkers for the diagnosis of Rett Syndrome.

Both knockdown and mutations in PIWIL1 induce defects in neuronal migration in the developing rat cerebral cortex (41). PIWIL1 knockdown also affects the multipolar–bipolar transition in post-mitotic neurons, which is essential for proper radial migration (41). Furthermore,PIWIL1 loss decreases the expression of microtubule-associated proteins such as Tau, MAP1B in the cortical tissues of adult mice. A whole exome sequencing study of families with simplex autism spectrum disorder identified strong correlations between de novo mutations in PIWI family members, particularly PIWIL2 and PIWIL4, and autism (42). Thus, PIWI family proteins likely regulate brain development, the dysregulation of which would likely be associated with autism and related disorders.

Gulf War Illness (GWI) is a chronic disorder suffered by 25% of veterans of the United States Army who were deployed to the Persian Gulf during the 1990–1991 Gulf War. Memory dysfunction and depression have been reported as the most prominent symptoms in brain and reduced volumes of cortical white and gray matters have also been reported (43.44). While the etiology of GWI is not completely understood, the ingestion of pyridostigmine bromide as a drug to prevent lethal nerve gas attacks and/or the overuse of insect repellants and pesticides such as N, N-diethyl-3-methyl-benzamide (DEET) and permethrin have been proposed. Epigenetic alterations were detected in a rat GWI model treated with pyridostigmine bromide, DEET and permethrin and exposed to mild stress, including altered methylation throughout the brain and altered expression of miRNAs and piRNAs (45). In particular, two piRNAs were upregulated in the hippocampus. The study of the GWI model implicates the involvement of non-coding RNAs and epigenetic alterations in the brain symptoms of GWI.

Associations of small non-coding RNAs with schizophrenia have also been analyzed in post-mortem samples of the anterior cingulate cortex from patients with schizophrenia. Although miRNAs and snoRNAs are differentially expressed between patients and controls, significant changes in piRNA expression were not detected in this study (46).

The findings of the aforementioned studies are summarized in Table 2, and the three most recent piRNA studies in subjects with ALS, AD and PD will be described below.

ALS is a progressive motor neuron disease. In degenerating neurons, the aggregation of RNA-binding proteins such as FUS and TDP-43 is observed in cell bodies (47.48.49). In normal cells, the majority of FUS and TDP-43 is physiologically localized in the nucleus and function in RNA metabolism (50.51). In Drosophila, neuron-specific knockdown of Cabeza( Caz,an ortholog of FUS), induce movement disorders accompanied by congenital defects of the neuromuscular junctions in motor neurons, suggesting that ALS pathology is partially recapitulated (52). In this fly model of ALS, long non-cording RNAs genetically and physically interact with Caz (53). Moreover, Caz genetically interacts with piRNA-associated genes, Aub, Ago3, Wde and Rhino (15). The Caz protein binds to transcribed pre-piRNAs and suppresses pre-piRNA expression in the CNS (15). Neuronal overexpression of Aub along withCaz knockdown causes morphological defects in neuromuscular junctions and locomotor deficits in Drosophila larvae (15). Increased levels of aberrant pre-piRNAs induced by Cazinactivation accumulate in the cytoplasm with the Caz protein, which is mediated by overexpressed Aub (15) (Figure 2). Because mRNA promotes FUS multimerization (54), abnormal pre-piRNAs might stimulate Caz aggregation with Aub in the cytoplasm, which may contribute to ALS pathogenesis.

Figure 2.

RNA-protein aggregation induced by aberrant pre-piRNAs in the Cazknockdown (ALS model) fly. (Left panel) In normal flies, Caz negatively regulates pre-piRNA expression and splices pre-piRNA transcripts in the nuclei. Processed pre-piRNAs are then exported by Caz to the cytoplasm to produce mature piRNAs. (Right panel) Caz suppression causes overproduction of pre-piRNAs, leading to the aggregation of RNA-protein complexes containing the remaining Caz protein in the cytoplasm, a process requiring the involvement of Aub (15).

Since abnormal pre-piRNAs and PIWI proteins have been proposed to induce the formation of RNA-protein aggregates, including FUS and TDP-43, thorough analyses of piRNA pathways in motor neurons from patients with ALS are warranted.

AD is the most common cause of dementia and is characterized by symptoms such as deficits in memory, learning and behavioral and social skills. Tau mutations are linked to AD, and neuronal Tau inclusions are thought to be a direct cause of neuron death in the hippocampus. Tau aggregation induces the decondensation of heterochromatin and depletion of Piwi in subjects with AD (55.16), allowing the activation of TEs in patients with AD (Figure 3). The combination of somatic pre-piRNA-encoding flamenco mutations and pathogenic Tau^R406W expression leads to aberrant cell cycle activation and neuronal cell death in Drosophila (16). Moreover, TEs are differentially expressed in the postmortem brain tissues from patients with AD and tauopathy-associated progressive supranuclear palsy. Additionally, dietary restriction and the administration of lamivudine, an inhibitor of reverse transcriptase, suppress the dysregulation of TEs and neurotoxicity induced by pathogenic tau expression in Drosophila (16).

Figure 3.

Pathogenic Tau-mediated dysregulation of the Piwi pathway in subjects with AD. Tau aggregation induces piRNA depletion and chromatin decondensation, which results in the ectopic transposition of TEs, leading to neuronal death through the ectopic activation of the cell cycle (16).

TE activation has also been implicated in motor neuron degeneration in subjects with ALS (56.57). Associations of piRNAs with AD have also reported in recent two studies (58.59). Further studies are needed to address the mechanism by which Tau aggregation induces chromatin and piRNA changes, as well as their importance in AD pathogenesis.

PD is the second most common ageing-associated neurodegenerative disorder and affects motor and non-motor neurons. The loss of dopaminergic neurons, which underlies the motor symptoms in patients with PD, is believed to be caused by mitochondrial dysfunction and/or the aggregation of the presynaptic protein /-synuclein.

The expression of piRNAs derived from short interspersed nuclear elements (SINEs) and LINEs is significantly downregulated in fibroblasts, induced pluripotent stem cells (iPSCs) and differentiated neuronal cells from patients with PD (17). The group of upregulated piRNAs in neurons differentiated from iPSCs and brain tissues was characterized by an increase in the cytosine content within the first 2-9 bp in patients with PD, although the pathological significance is unclear. Different levels of dysregulation of piRNAs have been observed in different cell types and differentiation stages (17). CpG methylation also differs among cell types, although differences have not been observed between patients and controls (17).

piRNAs have possible roles in methylating the promoter region of the CREB2 gene in Aplysia neurons, which modulates synaptic plasticity during memory formation (34). Downregulation of CREB pathway genes has also been detected in patients with PD, implying a possible connection between piRNA pathways and CREB inactivation by epigenetic modifications.