2.1 Classification of neurodegenerative diseases

Several criteria, including clinical symptoms, anatomical region of neuronal dysfunction, and major molecular or protein conformational variants, had been utilized to classify neurodegenerative diseases^{1, 14, 15} (Table 1). For example, based on clinical symptoms, neurodegenerative diseases could be classified as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and amyotrophic lateral sclerosis (ALS). In addition, neurodegenerative diseases can also be named amyloidosis, tauopathy, alpha-synucleopathy, and transactivation response DNA-binding protein 43 (TDP-43) proteinopathy according to the major molecular or protein conformational variants. These major molecules and proteins have specific conformational properties that influence neurodegenerative diseases.

2.2 Aging and neurodegenerative diseases

Aging refers to a series of degenerative changes that occur in tissues, organs, and the whole body over time as the body matures and cell function gradually declines and eventually dies.^{16, 17} Various pathogenic factors of neurodegenerative diseases were closely related to the aging process, such as abnormal deposition of proteins, DNA damage, mitochondrial dysfunction, cellular aging, metabolic dysfunction, dysregulation of nicotinamide adenine dinucleotide (NAD+) levels, oxidative stress, stress response, telomerase inactivation, and inflammation.¹⁸ Some of these features have been observed in AD and PD.^{18, 19} Then, exogenous administration of NAD+ could prolong the lifespan of C. elegans and improve the pathogenesis and pathological characteristics of age-related neurodegenerative diseases.²⁰ Furthermore, the inhibition of mTOR signaling by rapamycin enhanced neuroprotection and inhibited cellular senescence.²¹ Therefore, exploring the link between neurodegenerative diseases and aging characteristics may lead to new therapeutic strategies for such diseases.

2.3 Abnormal deposition of proteins

Neurodegenerative diseases are characterized by progressive damage and selective dysfunction of neurons, associated with pathologically altered proteins deposited in the human brain as well as in peripheral organs. Abnormal conformational protein deposition impaired normal physiological processes of the neuronal system.²² These proteins included the β-amyloid protein (Aβ), tauopathies, synuclein-alpha (SNCA), and TDP-43.²³ The deposition of abnormal proteins affected the function of neurons in brain tissue to varying degrees, resulting in cognitive and functional impairments²⁴ (Figure 1). Although biochemical modification of proteins is a potential therapeutic target and biomarker in neurodegenerative diseases, there is currently no effective clinical method for accurately identifying inclusion bodies formed by abnormal proteins in the course of neurodegenerative diseases.

3.1 Function of circRNAs

CircRNAs are a class of novel noncoding RNAs that are covalent closed-loop structures without the 5′ caps or 3′ poly (A) tails that linear RNAs possess, forming a continuous ring structure through covalent bonds.²⁵ CircRNA molecules have three cyclization patterns in different organisms: exon-skipping or lariat-driven circularization, direct back-splicing or intron-pairing-driven circularization, and RNA-binding protein-driven circularization^{26, 27} (Figure 2A). CircRNAs were highly abundantly expressed in the brain⁸ and involved in the regulation of biological processes through various regulatory mechanisms. For example, circRNAs regulated the expression of target genes through microRNAs (miRNAs) (Figure 2B). Previous studies have found that circRNA antisense to the cerebellar degeneration-related protein 1 transcript (CDR1as) had a powerful miRNA sponge effect and multiple miRNA-binding sites, such as miR-7, miR-135a, and miR-876-5p.^28-30 CircRNAs could also interact with specific RNA-binding proteins, thereby affecting the expression of related proteins (Figure 2C). CircRNA muscleblind (circMbl) was generated by circularization of the second exon of the Drosophila mbl gene, which in turn regulated circMbl levels by binding to the Mbl produced by its native mbl gene.³¹ Another circRNA glutamate ionotropic receptor AMPA type subunit 1 (circGRIA1) was abundantly expressed in the brain and could bind to glutamate receptor 1 to regulate synaptic plasticity and improve age-related synaptic function.³² Furthermore, circRNAs play important roles in cell biology as transcriptional regulators (Figure 2D). For example, circRNA human antigen R (circHuR) interacted with CCHC-type zinc finger nucleic acid binding (CNBP) to inhibit the binding of CNBP to the HuR promoter, thereby downregulating HuR expression levels and inhibiting the pathological process of gastric cancer.³³ Finally, although circRNAs were noncoding proteins, some recently discovered circRNAs function as coding proteins (Figure 2E). CircRNA zinc finger protein 609 (circ-ZNF609), which encoded a protein, had an open reading frame, and the basic elements of start and stop codons were the same as those of linear transcripts.³⁴ In summary, circRNAs mainly function by acting as miRNA sponges, regulating transcription, binding proteins, and translating polypeptides. CircRNAs, as a research hotspot in the field of ncRNAs, will reveal more important functions and mechanisms with the development of new technologies in the future.

3.2 Expression patterns of circRNAs in neurodegenerative diseases

CircRNAs are involved in the regulation of synaptic function, and their expression is age-related and tissue-specific. CircRNAs had a high expression level in the mammalian brain and accumulated in neuronal tissue with age,^{8, 35, 36} which might be due to their high stability.³⁷ CircRNAs were highly conserved and specifically expressed in mouse, human, and Drosophila brain tissues.^{8, 36} Eighty percent of all circRNAs in mouse neurons with high expression levels were detected in the human brain.⁸ CircRNAs in the Drosophila brain were the most abundant among all tissues.³⁶ The above results show that circRNAs have similar expression patterns in the brains of different model organisms, suggesting that circRNAs can stably exist in brain development and neuronal systems and can be used as biomarkers for brain pathologies.

Highly enriched circRNAs in the brain have extremely important roles in a variety of neurodegenerative diseases. Several dysregulated circRNAs have been identified in neurodegenerative diseases (Table 2). For example, 344 dysregulated circRNAs were detected in the brains of 6-month-old AD mice, of which 192 were upregulated and 152 were downregulated, and there were 244 dysregulated circRNAs in the brains of 9-month-old AD mice, of which 142 were upregulated and 102 were downregulated.³⁸ CircRNAs are abundant and dynamically expressed, suggesting their role in neurodevelopment as well as in the pathogenesis and progression of neurological diseases.

4.1 CircRNAs in AD

AD is a neurodegenerative disease typically of insidious onset and characterized by the presence of neurotic plaques and neurofibrillary tangles.⁴³ AD had two basic pathological features: (1) interneuronal neurofibrillary tangles composed of abnormally modified tauopathies and (2) accumulation of Aβ and formation of senile plaque deposits.⁴⁴ In addition, inclusion bodies of TDP-43 protein had also been found in AD patients.^{45, 46} To date, several circRNAs have been found to play a role in the regulation of AD pathological processes (Figure 3).

4.2 CircRNAs in Parkinson's disease

PD was a common progressive neurodegenerative disorder that was characterized by tremors and bradykinesia.⁶⁶ The main pathological feature of PD was the degeneration of the Lewy body or the Lewy nerve synapses due to SNCA aggregation, resulting in the formation of filamentous cytoplasmic inclusions, accompanied by lesions in the substantia nigra and dysregulation of dopamine homeostasis.⁶⁷ Most circRNAs regulate PD through a miRNA mechanism (Figure 4).

4.3 CircRNAs in HD

HD is a dominantly inherited neurodegenerative disease caused by repeated expansion of the CAG nucleotide sequence in the Huntington gene on chromosome 4, resulting in abnormal gene expression. Mutant huntingtin (mHTT) produced abnormally long polyglutamine repeats.⁸³ Twenty-six or fewer repeat amplifications of CAG were considered normal, whereas 27–35 replicates posed a risk of spreading diseases secondary to dilation through parental meiosis. Furthermore, 36–39 replicated CAG represented incomplete penetrance of HD pathology peaks, whereas CAG-expanded alleles beyond 40 repeats were considered fully penetrant genes and caused HD.⁸⁴ More CAG repeat expansions were generally associated with an earlier age of onset, and male sperm had a greater potential for repeat variation. Therefore, HD was often associated with genetic predisposition in men.^{85, 86} In addition, mHTT proteins lead to neuronal dysfunction and death through various mechanisms, such as the regulation of cellular protein stabilization, axonal transport, transcription, translation, and mitochondrial and synaptic functions.^87-89

One study found 19 downregulated and 4 upregulated circRNAs between mouse PC12 cell lines expressing wild-type huntingtin protein as a control and mHTT protein.⁹⁰ In this study, 16 downregulated circRNAs came from the same chromosomal region of the Rere gene, and the remaining three downregulated circRNAs came from other chromosomal regions.⁹⁰ From the analysis of the mechanism, 15 of the 23 differentially expressed circRNAs were related to the MAPK pathway, and 16 were involved in the dopaminergic synaptic pathway. The pathological mechanism of dopamine in HD has been widely demonstrated.⁹⁰ The MAPK pathway had important effects on cell proliferation and division, the stress response, differentiation, and apoptosis; c-Jun N-terminal kinase (JNK) and p38 in the MAPK pathway were the main signaling factors involved in the pathogenesis of HD.⁹¹ In summary, circRNAs might regulate the pathological process of HD through dopaminergic synapses and MAPK pathways, and their specific biological functions require further exploration.

Another study found that circHTT 2–6 derived from the exons 2, 3, 4, 5, and 6 of the HTT gene was enriched in in the frontal cortex of HD patients.⁹² When circHTT (2–6) was overexpressed in HEK293 and SH-SY5Y cells, no change in the CAG repeat region of HD was detected, which decreased the cell proliferation, nuclear area, and altered subcellular localization of the HTT protein.⁹² These results demonstrate the overexpression of circHTT undergoes HD-related pathological changes, but its specific functional mechanism needs further study.

4.4 CircRNAs in ALS

ALS is a heterogeneous neurodegenerative disease whose main pathological features are the degeneration of upper motor neurons that project to neurons in the brainstem and spinal cord and lower motor neurons that the brainstem or spinal cord projects onto muscles. ALS patients had features of TDP-43 proteinopathy, such as loss of TDP-43 in the nuclei of neuronal cells and cytoplasmic aggregated with skeletal-like or dense morphology in residual motor neurons.⁹³

In addition, circRNAs have been involved in the regulation of fused in sarcoma (FUS) proteins during ALS.⁹⁴ For example, circ-Hdgfrp3 took part in protecting neuronal function and integrity in neuronal cells, whereas mutant FUS protein (mtFUS) affected the localization of circ-Hdgfrp3 under oxidative stress conditions.95 The mtFUS could lead to abnormal accumulation of cytoplasmic FUS protein and increased mitochondrial translocation, resulting in excessive mitochondrial fission and damage, eventually leading to neuronal death, which was a major pathological feature in some ALS patients.¹² In summary, circRNAs are likely to be involved in the pathological regulation of ALS.

Many dysregulated circRNAs were also found in ALS patients, including 151 downregulated circRNAs, most of which were involved in the pathological process of ALS.⁹⁶ Hsa_circ_0000567 derived from the SETD3 gene, and SETD3 was the histone methyltransferase that regulated muscle differentiation in mouse.⁹⁷ Hsa_circ_0023919 originated from the PICALM gene, which was involved in clathrin-mediated endocytosis at neuromuscular junctions. Single-nucleotide polymorphisms upstream of the gene were associated with AD.⁹⁸ Simultaneously, the authors evaluated the correlation between the expression levels of circRNAs and the potential association with clinical data.⁹⁶ Three circRNAs (hsa_circ_0000567, hsa_circ_0023919, and hsa_circ_0088036) were negatively correlated with patient age at the time of blood collection.⁹⁶ The sensitivity and specificity of these three circRNAs were as high as 90% in patients with ALS, significantly higher than the most representative biomarkers in ALS, phosphorylated neurofilament heavy chain and neurofilament light chain.⁹⁶ Therefore, circRNAs have great potential as biomarkers for ALS diagnosis and treatment.