Pain of the orofacial region is the primary complaint for which patients seek treatment. Of all the orofacial pain conditions, one condition that possess a significant global health problem is temporomandibular disorder (TMD). Patients with TMD typically frequently complaints of pain as a symptom. TMD can occur due to complex interplay between peripheral and central sensitization, endogenous modulatory pathways, and cortical processing. For diagnosis of TMD pain a descriptive history, clinical assessment, and imaging is needed. However, due to the complex nature of pain an additional step is needed to render a definitive TMD diagnosis. In this review we explicate the role of different biomarkers involved in painful TMD. In painful TMD conditions, the role of biomarkers is still elusive. We believe that the identification of biomarkers associated with painful TMD may stimulate researchers and clinician to understand the mechanism underlying the pathogenesis of TMD and help them in developing newer methods for the diagnosis and management of TMD. Therefore, to understand the potential relationship of biomarkers, and painful TMD we categorize the biomarkers as molecular biomarkers, neuroimaging biomarkers and sensory biomarkers. In addition, we will briefly discuss pain genetics and the role of potential microRNA (miRNA) involved in TMD pain. The orofacial region consists of heterogeneous hard and soft tissues that make diagnosing and treating pain conditions a challenging task. Pain of the orofacial region is debilitating and can arise from various structures innervated by the cranial nerves in particular trigeminal nerve. Majority of the orofacial structures transmit impulses through trigeminal pathways to the brain where pain is perceived as a subjective sensation by the dynamic interaction of cognitive, affective and sensory elements. Orofacial pain is a multidimensional experience that can affect the patient physically, emotionally, and impairs quality of life. The conditions associated with orofacial pain are broadly divided into different categories such as odontogenic, musculoskeletal, neurogenic, psychologic, headaches, and others. Clinically, due to diverse nature of pain complaints, it is difficult to distinguish many of the orofacial pain disorders that eventually leads to misdiagnosis, delay in diagnosis or ineffective management. It becomes more challenging for the clinician when the pain is persistent and devoid of any physical and or structural cause.3 Women are more frequently affected by a ratio of 2:1, and other described risk factors are low socioeconomic status, smoking, psychological factors and presence of other chronic pain conditions.5Recently, population-based cross-sectional surveys have shown that the prevalence rate of self-reported orofacial pain ranges from 10 to 26% and cost over 32 billion each year in the United States.6In clinical settings, pain conditions are categorized as acute or chronic. In most instances, definition of chronicity varies among researcher and health care practitioner. According to the International Association for the Study of Pain “chronic” indicate persistence of pain beyond the normal time of healing, while for clinical practicability a duration exceeding 3 months and for research purposes, a duration of 6 months is used to denote the pain as being chronic.Acute pain is an adaptive, self-limited involving activation of nociceptors and chemical mediators with clearly defined cause. It may be very intense, often accompanied by anxiety and restlessness but the cause-and-effect relationship is usually apparent and treatment measures are usually effective. While chronic pain is persistent and resistant to treatment measures because of neuroplastic changes in the structures of central and peripheral nervous system. Presently, prevalence of chronic pain is increasing due to changes in work demands and increased symptom awareness and turn out to be major source of suffering as it interferes with daily functioning accompanied by distress, hopelessness, fatigue, depression and dependence. One of the chronic pain conditions which possess a global health problem is temporomandibular disorders (TMD).7 TMD affects about 5%–12% of the general population,7 while some studies9 reported higher incidence of TMD as 25%–40% with women having greater risk of developing TMD conditions than men.7 It is the second most common musculoskeletal pain condition after chronic back pain.TMD are defined as a group of clinical conditions affecting the muscles of mastication, the temporomandibular joint (TMJ), and the related structures.10 In this review, we have used the term “painful TMD” which includes both primary and secondary pain disorders.Of all the painful orofacial conditions, most common conditions for which patient seek treatment are tooth-related pain and painful TMD. According to the recent classification on Orofacial Pain, temporomandibular joint pain (TMJ) indicates pain localized to the TMJ, occurring at rest or during jaw movement or palpation. TMJ pain is classified as primary pain which means pain that is not attributable to another disorder and secondary pain means pain caused by another identified disorder such as inflammation or due to infection, trauma, or autoimmune disorder, sensitization of the tissues, structural changes, muscle spasm, or injury. Secondary TMJ pain is diagnosed usually when the pain has developed in temporal relation to onset or substantial worsening of the presumed causative disorder or has led to its discovery or the pain has significantly worsened in parallel with progression of the presumed causative disorder which can be either disc disorders or degenerative joint diseases (DJD).11 Genetic and sensory processing also contributes to the etiology of TMD. Recently, the Orofacial Pain Prospective and Risk Assessment (OPPERA) study reported that TMD is best viewed using a biopsychosocial model to emphasize the importance of psychosocial and behavioral factors to the onset of painful TMD.12Painful TMD potentially occurs due to ongoing nociceptive input and interactions between peripheral and central sensitization, endogenous modulatory pathways, cortical processing and psychologic factors. Patient with TMD frequently report pain as the primary complaint that can occur from either intra-articular structures or from an extra-articular location as referred pain from adjacent structures. Other symptoms of TMD included joint noises, impaired jaw movements tinnitus, dizziness, cervical pain and headaches. TMD has multifactorial etiologies and consists of various contributing factors including trauma, either physical or emotional, biological process such as aging, postural condition such as abnormal head and cervical position, systemic predisposition, sleep disorders and psychosocial alterations.13 Further, epidemiological studies show that comorbidities are common among individuals with TMD pain. It includes headaches, widespread pain and fibromyalgia, neck and back pain, the most common psychosocial disturbances such as stress, anxiety, depression and pain catastrophe and other visceral comorbid conditions such as irritable bowel syndrome and pelvic pain.15 These comorbidities not only co-occur with TMD but also contribute to onset and persistence of pain. Therefore, for diagnosis of painful TMD conditions, a special attention should be given to the history and clinical assessment. A descriptive history is required to render TMD diagnoses and identify the contributing factors. In some cases, clinical examination and history are not reliable and provide limited diagnostic information.16 So, diagnostic imaging is needed, in some cases to render a TMD diagnosis and to rule out other pathology. Moreover, several issues have emerged at the diagnostic level of TMD due to heterogeneity of the disorder. Also, association of multiple pain conditions such as fibromyalgia and back pain with TMD in a single patient or presentation of single pain complaint in multiple ways like referral pain in TMD and other orofacial pain conditions make this disorder more complex.15 Also, the underlying mechanism differ in each pain conditions and it is hard to identify risk factors in painful TMD as symptom of pain varies with time that depends on the chronicity of the condition. Additionally, differences in the TMD pain between the individuals at diverse points of time contribute to more challenges in diagnosis as well as in management of the TMD. Due to this inherent complexity of painful TMD and poorly understood etiologies making diagnoses and subsequent treatments exceedingly difficult. With a significant proportion of the population suffering from painful TMD, the development of new and accurate diagnostic procedures is essential to improve the current standard of care.  and matrix metalloproteinases (MMP) are common biomarkers involved in individuals with painful TMD.28 However, it should be noted that an ideal biomarker in painful TMD should have high sensitivity and specificity and can easily be quantified before obvious clinical symptoms are present in patients and during or after treatment.Cytokines are small peptides that are released during inflammation and have both pro- and anti-inflammatory effects (proinflammatory biomarkers Interleukin 1 beta (IL- 1β), IL-6, TNF, and IL-8 and anti-inflammatory biomarkers IL-4 and IL-10). Since these cytokines are studied in inflammation their potential role in pain secondary to disc displacements, DJD and muscle disorders have gained considerable interest.29 There are studies that have investigated the potential role of IL in which pain occurs secondary to DJD, disc, and muscle disorders. In a review, the synovial fluid was analyzed in patients with painful DJD. Researchers observed that increased cytokines, IL-1β typically result in the development and progression of painful TMD.31 In contrast, some studies reported high synovial fluid IL-1ra and low IL-1β concentrations in patients with resolution of arthritis.32 In a study Ogura et al., noticed that IL-1β upregulate monocyte chemoattractant protein-1 (MCP-1) in serum and saliva which is chemokine and considered to be involved in recruitment of monocytes to the TMJ synovium in degenerative joint diseases patients.33 Also, MCP-1 is expressed in sensory neurons, and its expression in both neurons and peripheral tissues is upregulated following local inflammation.33To date, three subtypes of IL-1 have been identified such as inflammatory IL-1α and IL-1β, as well as IL-1 receptor antagonist, IL-1ra. IL-1 induces several inflammatory events and plays a vital role in hyperalgesia through activation of lymphocytes, stimulation of other cytokines, prostaglandin, and collagenase release from connective tissue cells. IL-1 family have a significant impact on TMJ homeostasis.34 Similarly, studies36 have shown a relationship between inflammatory process and destruction of TMJ synovium and IL-6 has been pointed out as one of the most important proinflammatory cytokines contributing to the pathogenesis of disc disorder and degenerative changes. IL-6 is a pleiotropic cytokine produced by produced by variety of cells such as synovial cells, endothelial cells, macrophages, T-cells, smooth muscle cells, and fibroblast in the TMJ. It plays an important role in transition from acute to chronic pain inflammation and presents a dual effect with both anti and proinflammatory effect. In a study, level of IL-6 was observed higher in the synovial fluid of patients with TMJ pain with DJD compared to healthy controls which states that high IL-6 levels are associated with pain.36 It is noteworthy that IL-8 is another proinflammatory cytokine consisting of multifunctional actions in severe and critical inflammation.37 Sato et al., also found a high association between the levels of IL-6 and IL-8 in the synovial fluid of the patients with disc disorders and degenerative changes, indicating that a collaborative activation of theses cytokines is possible in TMD.38 In addition to synovial fluid analysis, intra-muscular cytokines and salivary biomarkers have also been studied in TMD. In a study, an elevated level of IL-6, IL-7, IL-8, and IL-13 have been observed in the masseter muscles of patients with TMD myalgia.39Likewise, Slade et al. observed high concentration of IL-1 and IL-8 in serum and saliva in patients with widespread pain and TMD.40In another study, Slade et al. observed the role of cytokines MCP-1, IL-1ra, and IL-8 in TMD with and without widespread pain. They observed odds ratios of 1.5 for MCP-1 and 1.4 for IL-1ra which signified that an increase in concentration of each cytokine was associated with an increase of ~50% in odds of TMD without widespread pain. Also, high levels of MCP-1 and IL-8 increased odds of TMD with widespread pain, with no independent contribution of IL-1ra was observed. They concluded that TMD without widespread pain cases had elevated levels of the proinflammatory cytokine MCP-1 and the anti-inflammatory cytokine IL-1ra. In contrast, TMD with widespread cases had elevated levels of the proinflammatory cytokine IL-8 with no compensatory increases in IL-1ra, thus indicating a more severe pro-anti-inflammatory imbalance among TMD with widespread cases.Overall, above cross-sectional and case-control studies showed that pain in TMJ is associated with inflammation secondary to arthritis or disc disorders and molecular markers such as IL-1β, IL-6, and IL-8 plays an important role in the onset and progression of the disease. These markers can be used to predict the outcome and progression of painful TMD. These studies also reported that IL-1β, IL-6, and IL-8 have high sensitivity and high specificity to painful TMJ with underlying inflammation. However, it is still unclear which specific markers needed to be assayed.23 Interestingly, high concentrations of TNF also observed in dorsal root ganglion neurons and peripheral afferent fibers which explains its specific role in the peripheral mechanism of pain.42TNF is synthesized by many cells such as macrophages and monocytes, T cells, B cells, and fibroblasts. TNF can also upregulate its own synthesis and rapidly induces synthesis of other mediators such as IL-1, IL-6, and prostaglandins. Conversely, several cytokines such as endogenous immunosuppressive cytokines IL-4 and IL-10 and inhibitors of prostaglandin synthesis and glucocorticoids downregulate TNF expression.43 Nordahl et al., reported that the patients with chronic inflammatory connective tissue disease and associated TMJ pain have elevated TNF levels in the synovial fluid compared to those without pain.44 Also, in a clinical study a high pretreatment level of TNF in the TMJ synovial fluid was found to be associated with TMJ pain. However, after treatment with glucocorticoid intraarticular injection, a reduction of synovial fluid TNF and pain relief was observed.45 These studies explain that TNF has high sensitivity in inflammatory diseases and pain that can be used as a marker to understand the peripheral mechanism of pain and to assess the progression of painful TMJ disorders.In a study, synovial fluid TNF levels have been shown to be significantly higher in individuals with TMJ pain upon mandibular movement than in those without such pain.24Based on the discussion of IL and TNF it could be considered that underlying inflammation in patients with TMD is one of the potential sources of pain. Another family of cytokines called MMPs are discussed here. MMPs are group of enzymes that are released during inflammation and associated with tissue destruction. MMPs are proteolytic enzymes that degrade extracellular matrix components and has been noticed to be involved in TMJ inflammation and pain modulation.48 Also, in few other studies, in patients with disc disorders and degenerative joint changes an increased level of collagenases , Stromelysin (MMP-3), and gelatinases (MMP-2 and MMP-7) in TMJ synovial fluid was observed.53After exploring the literature, it has been noticed that the expression of MMP-1, MMP-2, and MMP-13 increases in joint inflammation which contributes to progressing of degenerative changes and pain.54 Also, Nascimento et al., analyzed the expression of MMP-2 and MMP-9 in the rat trigeminal ganglion during the development of TMJ inflammation.55 In this study researchers evaluated whether mechanical allodynia orofacial hyperalgesia, induced by the injection of complete Freund’s adjuvant into the TMJ capsule, were altered by an MMP inhibitor . They observed MMP ex-pression in the trigeminal ganglion was shown to vary during the phases of the inflammatory process. MMP-9 regulated the early phase and MMP-2 participated in the late phase of inflammatory process. Additionally, the increases of mechanical allodynia and orofacial hyperalgesia were attenuated by the with DOX a nonspecific MMP inhibitor.52 In painful TMD research, MMPs are gaining a considerable interest and it can be used as a therapeutic target for management of pain. Although multiple molecular biomarkers have found to be above than normal concentrations in painful TMD, it is still unclear how specific mediators should be chosen. Also, the concentrations of cytokines vary considerably in individuals with painful TMD. Therefore, multiple biomarkers should be assayed to understand the complexity of painful TMD.In pre-clinical models Huang X et al., observe the effect of psychological stress on TMJ of rats. They noticed an increased expression of MMP-3 correlated well with degree of TMJ lesion.56 Similarly, prostaglandin PGE2 produced by COX-enzyme during inflammation is also an important biomarker observed in pain studies and found to associated with in delayed onset muscle soreness.23 In addition, Alstergren et al., also detected increased levels of PGE2 in synovial fluid of patients with chronic inflammatory joint disease, and the levels were found to be related to TMJ pain on mandibular movement.57 Recently Basi et al., studied the biomarkers in TMD patients and controls.58 They collected venous blood, biopsied masseter muscles, and TMJ synovial fluid on the subjects’ side of maximum pain intensity with disc displacement and concurrent joint pain and myofascial pain. They assayed bradykinin, nerve growth factor, PGE2, LTB4, F2-isoprostane, and substance P and noticed that individual pain mediators analyzed were not correlated between plasma, muscle, synovial fluid, and painful TMD except for F2-Isoprostane.58Bradykinin is one of the inflammatory mediators and plays an important role in nociception and sensitization. It is a potent vasodilator and bronchoconstrictor that increases vascular permeability and facilitates pain transmission. In a study on TMJ disc disorders and DJD high bradykinin levels were observed correlated positively with the degree of inflammationIn painful TMDs, neurotransmitters play a key role in peripheral or central sensitization as well as central processing. Along with pain processing neurotransmitter have strong impact on mood and behavior. All the major neurotransmitters are inflammatory mediators included PGE2, PGI2, LTB4, NGF, Bradykinin, adenosine, substance P, 5-HT, histamine, glutamate, noradrenalin, NO and non-inflammatory mediators as CGRP, GABA, opioid peptides, glycine, and cannabinoids that are typically involved in inhibitory and facilitatory pain mechanism. Currently, monoamine neurotransmitters such as glutamate, serotonin (5-HT), and dopamine have been explored as potential biomarkers in painful TMD conditions.59Glutamate is the most abundant excitatory neurotransmitter present in afferent sensory nerves for conveying sensory information to the central nervous system (CNS). It is present in the trigeminal ganglion as well as in the central and peripheral inflammatory sites. Typically, the sensory neurons contain full set of glutamate receptors including NMDA (N-methyl-D-aspartate), AMPA (α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate) which are the potent modulators of central sensitization.61 This finding suggest that glutamate can arise from nerve fibers in the inflamed synovial membrane or from plasma extravasation into the synovial tissues and can be used to understand the underlying mechanism of peripheral and central sensitization specifically in patients with painful TMD. This also means that glutamate has high sensitivity and specificity towards painful TMD. It is also noticed that glutamate injection in the healthy TMJ evokes immediate pain that is partly mediated by peripheral NMDA receptors in the synovial tissues. In order to overcome TMJ pain, researchers tried to peripherally block the NMDA receptor by ketamine or NMDA antagonist and noticed partial reduction in pain.61 This observation explains the role of glutamate in pain processing. However, additional studies are needed to understand the role of glutamate in pain behavior in painful TMDs.In a study, researchers noticed an elevated level of glutamate in the joints with inflammatory conditions such as degenerative diseases secondary to rheumatoid arthritis. They reported that glutamate usually act as a mediator and is not primarily associated with inflammation whilst its receptors have been found to possess modulatory roles in peripheral nociception and sensitization.62Serotonin or 5-hydroxytryptamine (5-HT) modulates physiological processes in both the central and peripheral nervous systems. It is synthesized both peripherally and in the CNS from the essential amino acid tryptophan, which is derived from the diet. In the CNS, 5-HT is found in serotonergic neurons and considered to have an important role in control of pain through descending inhibition. However, outside of CNS 5-HT act as an inflammatory mediator and sensitizes afferent nerve fibers to induce hyperalgesia.64 Previous research noticed an increase number of 5-HT3 receptors on sensory nerves of the masseter muscle in patients with myalgia compared to controls which showed that serotonin is involved in in myalgia.66 5-HT also sensitizes peripheral mechanoreceptive afferent fibers to other chemicals such as, glutamate, Substance P (SP), Calcitonin gene related peptide (CGRP) by enhancing the efficiency of tetrodotoxin-resistant sodium channels and lowering the threshold of TRPV1 receptors, which results in primary hyperalgesia.67 This explains that in patients with hyperalgesia serotonin can be used a biomarker. In TMJ of healthy individuals’ serotonin is usually undetectable in synovial fluid which is important from a diagnostic point of view. In a study, investigators observed an elevated serotonin levels in the TMJ synovial fluid that has been strongly associated with TMJ pain provoked by functional mandibular movements.65 Several other studies have demonstrated that patients with chronic myalgia and myofascial pain have significantly higher level of 5-HT in saliva and serum compared to control and high levels are correlated with allodynia.67 In another study, in patients with fibromyalgia, elevated synovial 5-HT has been observed to be associated with increased pain and high levels of anxiety.66 In another clinical study, researchers investigated the influence of 5-HT on the effects of intra articular injections of glucocorticoid on pain of the TMJ in patients with inflammatory disorders of TMJ. Researchers concluded that both local and systemic levels of 5-HT partly determine the effect of intra-articular glucocorticoid injection on TMJ pain in patients with chronic arthritis or degenerative diseases.68 From the above-mentioned studies, it could be inferred that serotonin plays a vital role in in peripheral and central pain mechanism and highly predictive of painful TMD compared to other biomarkers.It is observed that in response to tissue trauma and inflammation, 5-HT3 a subtype of 5-HT is released and sensitizes peripheral neurons which seems to be important for mediating pain from the periphery.69 These findings suggest that peripheral dopamine might be involved in modulating peripheral pain and a complex relationship existed between the peripheral and central actions of these neurotransmitters in TMD pain.In addition to 5-HT, Dopamine is also involved in pain perception as well as in motor control, cognition, and reward system. Primarily dopaminergic neurons synthesize dopamine in the CNS while adrenal medulla and neuroendocrine cells synthesize dopamine in the peripheral nervous system. A more recent study found no difference in plasma levels of 5-HT between patients with myofascial TMD and healthy controls, but that plasma dopamine levels were significantly increased in TMD patients.70Neuropeptides are small peptides that are released from neuronal cells and are commonly used by neurons to communicate with each other. Many neuropeptides are co-released with other neurotransmitters, such as glutamate, acetylcholine, and norepinephrine. Some neuropeptides are released peripherally and have a key role in neurogenic inflammation, such as SP, CGRP), etc. whereas others are released in central regions, such as galanin, cholecystokinin, and neurotensin. It is observed that CGRP and substance P are widely produced both in central and peripheral nervous system and can amplify variety of inflammatory and nociceptive processes.71 These observations suggest that SP levels may reflect joint injury or pathology, whereas CGRP levels may be more reflective of joint pain. However, it is not clear whether these associations reflect increased expression in response to mechanical joint injury, or whether these neuropeptides are secreted in response to local tissue hypoxia that may occur in TMD.73 Therefore, more studies are needed to elucidate the complex relationship between SP, CGRP levels and pain. In addition to synovial fluid as medium for analysis, saliva has also been employed as an indicator of stress and chronic pain. For example, reports state that substance P, a neuropeptide associated with inflammation and pain, the stress hormone cortisol, and markers of oxidative stress can be repeatedly detected within salivary secretions. Suggesting a preference for saliva over serum in the detection of select markers, researchers go on to describe that substance P is actually more readily available in oral fluids than patient-matched blood samples.In a study of painful TMD patients, CGRP levels are positively correlated with pain and SP have been found to be increased in synovial samples with no positive correlation.74 Additionally, NGF also produces hypersensitization and elevated NGF levels were found in the plasma of patients from persistent inflammatory pain states, such as bladder pain syndrome chronic prostatitis and chronic migraine. Moreover, increased NGF was found in synovial fluid of patients with DJD secondary to RA, and cancer pain.73The nerve growth factor (NGF) belongs to the family of neurotrophins, a well-known mediator for persistent pain. In general, NGF is expressed after the injury or inflammation to the TMJ, and initiates signal cascades at the peripheral sensory neurons. This cascade involves activation of the PI3K-AKT pathway and ERK-phosphorylation, as well as activation of the protein kinases PKCγ and PKA, leading to sensitization of ion channels, such as TRPV1 that subsequently enhanced nociceptive activity and inflammatory pain.75 Other factors such as brain-derived neurotrophic factor, fibroblast growth factor, insulin like growth factor-binding protein and transforming growth factor were detected in TMJs with disc disorders and degenerative joint diseases.24In addition, vascular endothelial growth factor (VEGF) produced by endothelial cells, chondrocytes and osteoclast have also found increased in condylar cartilage of mechanically induced DJD and in synovial fluid and tissue in patients with disc disorders associated with tissue degeneration.Although many studies have used synovial fluid for analysis, along with saliva and serum these biofluids still provide an invaluable source of biochemical information. Also, acquiring synovial samples is accompanied by a degree of invasiveness based on which it can be inferred that at this time there is no one ideal biofluid capable of imparting all aspects of the TMD. While serum, saliva, and synovial fluids all contain biomarkers indicative of unique disease states, both local and systemic, none are considered comprehensive, hence necessitating the ongoing research for personalized diagnostics and therapeutics.76 They bind to the 3′ untranslated region (UTR), or occasionally 5′ UTRs, of the multiple mRNA targets to which they exhibit imperfect, or sometimes, perfect complementarity. This enables one specific miRNA to inhibit the translation of multiple genes.77 Also, it is established that miRNAs are essential regulators of many proteins involved in proper neuronal function and takes part in the morphological and functional changes sustaining neuronal plasticity.77To understand the pain chronification one area which has drawn the attention of many researchers is epigenetics in chronic pain. It is noticed that microRNAs (miRNAs) a class of small non-coding inhibitory RNAs play an important role in regulating pain processing within a wide range of experimental models and clinical pain disorders. miRNA is a novel class of non-coding single-stranded RNA of 19–24 nucleotides with the ability to modulate a large proportion of the genome post-transcriptionally.78 In another study, researchers evaluated the miRNA 140-5p that is expressed in TMJ DJD. They suggested that miR-140-5p may play a role in regulating mandibular condylar cartilage homeostasis and potentially serve as a novel prognostic factor of TMJ degenerative changes.79 In other few studies the biological function of the micro–ribonucleic acid 101a‐3p (miR‐101a‐3p) and miR21-5p (MiR21) in TMJ degenerative changes were explored.80 Researchers suggested that both miR‐101a‐3p and miR21-5p was involved in the cartilage matrix degradation as well as in progression of degenerative changes of TMJ.81So far there are a few published studies evaluating these biomarker profiles in TMD. In a study, a decreased expression of the miRNA221–3p was observed in synovial fibroblasts from patients with degenerative joint diseases. The researchers found that miRNA221–3p inhibits transcription of Ets-1, which is itself a transcription factor for MMPs enzymes responsible for tissue degradation and re-modeling in the joint cartilage.From the above results, it could be suggested that further studies on miRNA are needed which produce the same results to understand the exact involvement of miRNA and painful TMD.84 Researchers observed that increased excitability in central nociceptive mediating neurons and centers associated with pain as well as decreased activity in the descending inhibitory system seems to be involved in process of painful TMD.85 From this observation it could be anticipated that there are certain areas in the brain that specifically show alterations in painful TMD and can be used as a Biomarkers. Therefore, to explore those biomarkers investigators have used an enticing method called neuroimaging.Although it is observed that peripheral and central mechanism are involved in the pathogenesis of painful TMD, the neurobiological mechanism pertaining to pain remains to be clarified.86 Also, one very interesting technique that has been recently applied to assess white matter as well as in grey matter volume in the brain of TMD patients is MRI diffusion tensor imaging/fractional anisotropy combines with sMRI. These techniques also have demonstrated changes in massive reorganization and functional connectivity of the spinal, subcortical and cortical structures involved in pain perception and modulation in patients with neuroplastic changes or chronic pain conditions like TMD.87Neuroimaging investigates the brain function, structures, and changes in neurochemicals which can be used as a potential biomarker in painful TMD. Functional and structural magnetic resonance imaging (fMRI) and (sMRI) and magnetic resonance spectroscopy (MRS) methods have been widely used separately or combined to explore brain alterations in patients with chronic pain.Neuroplasticity is thought to occur following continuous barrage of nociceptive afferent signals to the brain. Hence, TMD pain is presumed to induce anatomical and physiological changes in the brain over time. Inherent risk factors which give rise to onset of chronic pain, may also contribute to brain abnormalities present in patients. Therefore, in this paper, we are familiarizing the reader with studies that have investigated the neuroimaging biomarkers of painful TMD patients in different regions of brain such as ascending pain system, lateral-medial pain systems, default mode network and antinociceptive descending pain modulations system and motor system Fig. 87 Similarly, investigators also noticed an increased cerebral blood flow and decreased grey matter volume (GMV) in the ipsilateral spinal sub nucleus caudalis (SpVc) which processes nociceptive input of the TMD patients. They reported that reduction in grey matter volume reflect neuronal loss, while the elevated blood flow in SpVc could be a compensatory response of increased neural activity to structural reductions or hyperexcitability due to nociceptive pathways.88There are structural MRI studies that have compared TMD patients and controls and found evidence of decreased nerve fiber density, axonal diameter and myelination as well as microstructural changes in response to nociception in trigeminal nerve roots.88 This potentially due to persistent trigeminal nociceptive input, which further contributed to the hyperalgesia of TMD by enhanced facilitating trigeminal sensory information in thalamus. These findings suggest that micro structural changes of the nerve in form of fiber density and GMV can be used as biomarker to understand the complexity of painful TMD mechanism. However, it seems like these neuroimaging findings have low sensitivity and reproducibility so further studies are needed to validate these findings.89In another study, GMV has been assessed in thalamus. Investigators observed a positive correlation between thalamus GMV and pain duration.90 In another study using fMRI Zhang et al. found decreased regional homogeneity in the right anterior insula in female patients with TMJ synovitis pain relative to healthy controls.91 Zhang et al., also reported decreased functional connectivity (FC) between median cingulate cortex (MCC) and anterior insula as well as decreased FC between MCC and dorsolateral prefrontal cortex (DLPFC) which was negatively correlated with pain intensity in TMD patients, which means patients with decreased connectivity reported higher pain.92Also, there are studies that observed different cortical regions using fMRI. Investigators have studied lateral pain system which is involved in encoding the pain intensity as well as somatotopy. Also, medial pain system implicated in mediating the more affective-motivational aspects in the experience of pain have been evaluated. In a study Younger et al. investigated female patients with myofascial TMD and reported increased GMV in the right anterior insula and a negative correlation between self-reported pain intensity and GMV in the anterior cingulate cortex (ACC) and posterior cingulate cortex (PCC) were observed.93 In a study by Weissman-Fogel et al., observed a functional disconnection within the DMN of prefrontal cortex (mPFC) and posterior cingulate cortex (PCC) in TMD patients with emotional interference.94 From these studies it is implicated that mPFC and PCC are two functionally connected regions processing introspective thoughts which is required for coping TMD pain. Also, in another study pain rumination scores which means repetitive negative thinking about pain were found to be positively correlated with the FC between mPFC and PCC, and periaqueductal gray PAG.95 This implies that TMD pain alters the normal function of these circuits which affects pain perception. Further, a key region of the endogenous pain inhibitory system PAG is well-positioned to modulate pain perception for interactions between ascending inputs from peripheral tissue and descending projections from brain regions such as ACC and mPFC and shows anatomical alterations in TMD pain conditions.96 Wilcox et al., demonstrated that in TMD participants, the PAG displayed a significant increase of mean diffusivity (MD) value and no GMV change, while the nucleus raphe magnus showed GMV decrease and no change in diffusivity.87 In another study using sMRI to assess the cortical thickness. Researchers reported that TMD patients had thicker cortex in the frontal pole and ventrolateral prefrontal cortex compared with healthy controls, and that cortical thickness of orbitofrontal cortex was negatively correlated to pain unpleasantness88 while cortical thickness in the left ventromedial prefrontal cortex was positively correlated with neuroticism scores in TMD patients.97Furthermore, default mode network (DMN) is a group of functionally interconnected brain regions that get activated during mind wandering and not involved in any specific task and becomes correspondingly deactivated during goal-oriented tasks has been studied in previous studies.99 Wessman-Fogel et al., found that TMD patients showed elevated activity in the primary motor cortex (M1) and supplementary motor areas (SMA) during the cognitive interference.94 Furthermore, in an sMRI study by Salomons et al., observed self-reported helpless-ness in TMD patients assessed by Pain Catastrophizing Scale was positively correlated with the cortical thickness of SMA which is a critical region implicated in cognitive aspects of motor behavior.100 However, further analysis identified neither significant group difference in the cortical thickness of SMA nor correlation with pain characteristics.99Investigators have also assessed the motor system in patients with TMD pain.Overall, the observation that certain brain areas are activated by transient painful stimuli in TMD patients, and that pain intensity is used for grading the magnitude has prompted researchers to understand brain activity that could serve as biomarkers to measure pain objectively. However, most of the brain responses observed when pain is present can also be observed when pain is absent. For example, similar brain responses can be elicited by non-painful auditory, tactile and visual stimuli, and such responses can even be recorded in patients. Therefore, there is still disagreement on the degree to which current measures of brain activity exactly relate to TMD pain. Furthermore, whether more recent analysis techniques can be used to identify distributed patterns of brain activity specific for pain can be only warranted using carefully designed control conditions. Also, the clinical utility of current pain biomarkers derived from structural and functional neuroimaging appears to be overstated, and evidence for their efficacy in clinical conditions is scarce. Initial results were promising need to be further tested for replicability and generalizability.101 In these studies, MRS imaging was used to assess neurochemical metabolites. It includes are N-acetyl aspartate (NAA), choline (Cho), and total creatine (tCr) as well as other metabolites such as glutamate (Glu), glutamine (Gln), and myo-insitol.101 According to the previous literature NAA is a biomarker of neuronal health and axonal numbers, while Cho is associated with in-creased cell numbers, membrane synthesis, or membrane breakdown. tCr is considered an important marker of cell energy and cell metabolism. Glutamine is a metabolite of glutamate and together they participate in complex metabolic activity cycles and intercellular communication involving neurons and astrocytes.103 MI is primarily present in glial cells and plays an important role in osmoregulation.105 It is observed that few of these neurochemicals can be used to assess the underlying mechanism of TMD Pain.Several studies have indicated that TMD pain conditions are associated with changes in brain Metabolism.106 Similarly, Harfeldt et al. reported elevated tCr levels within the posterior insula in TMD patients relative to healthy controls. Likewise, in the same study, increased Cho levels correlated with a reduced capacity for mouth opening and lower pressure pain threshold on the hand, and Glu levels were positively correlated with temporal summation of the nociceptive mechanical stimulus.101 These findings provide new evidence about the involvement of the neurochemicals as a biomarker in the neurobiology of underlying TMD. It is also a further step towards understanding and accepting that biomarkers can be used to assess the pain mechanism. However, need to be further tested for replicability and validity. It is observed that few of these neurochemicals can be used to assess the underlying mechanism of TMD Pain.In a study, Gerstner et al., found a negative correlation between Gln levels in the left insula and pain in TMD patients while NAA and Cho levels in the left posterior insula were increased compared to healthy controls. In this study NAA levels were positively correlated with the duration of pain.107 Therefore, comprehensive psychophysical examination provides unique, sensitive, and specific information about the neurological deficits of neuropathy and neuropathic pain. These examination helps the clinician to quantify the function of nerves involved in pain transmission. One of the tests that detect the sensory functioning is quantitative sensory testing (QST). Quantitative sensory testing (QST) is a psychophysical test method that investigates the functional state of the somatosensory system of a patient with regard to the severity of clinical signs by means of calibrated stimuli and subjective perception thresholds.108 It determines normal and aberrant sensory parameters, such as mechanical, cold, or heat detection and pain thresholds in patients with orofacial symptoms.Although the investigators have been working on eliciting the potential biomarkers in TMD pain, still the somatosensory changes underlying this disorder are not fully understood. Previous studies indicate that somatosensory abnormalities can play a vital role in the underlying pain mechanism of chronic TMD.111 In addition, different somatosensory profile can also be identified in subgroups of myofascial pain TMD.112 In a study investigator reported alterations in the QST profiles of some TMD patients compared to healthy controls and chronic widespread fibromyalgia patients suggesting that QST may be able to provide specific pain signatures that could be used to assist in the diagnosis of painful TMD.113 In another QST study of TMD patients have found that enhanced pain sensitivity is associated with subsequent development of TMD, and that TMD is associated with various abnormalities in somatosensory profiles compared to controls.115 Recently, multiple phenotypes of facial somatosensory abnormalities were detected in Chinese TMD arthralgia patients using QST despite the disappearance of clinical signs and symptoms using QST.116 Although various QST measures have been correlated with subjective pain in the previous case-control study still there is a lack of evidence in the literature for the use of QST as a clinical predictor of TMD. Therefore, more research is required to explore the relationship between QST parameters in patients with TMD pain conditions.However, QST rarely used in clinical practice as it requires the active participation of the individual, and wide variations between the patients so, it lacks the validity. Yet, when carried out in a strictly standardized condition, this method is reliable to assess sensory nerve function. In TMD most frequent somatosensory abnormalities are gain of function  to pressure, pinprick, cold and heat stimuli, and an increased temporal summation.117 Thus, each step in the sequence from genetic code to gene expression is a potential source for biomarkers.In addition to major advances in the molecular and neuroimaging research, pain biomarkers have also been assessed using person’s genetic code called as pain genetics. It is reported that epigenetic regulation can modulate gene expression, altering gene function without disturbing the underlying genetic code.119 As these neurotransmitters play important roles in processing nociceptive inputs, this gene has been the focus of a wide range of studies of diverse acute, inflammatory, and chronic TMD pain. COMT is expressed in several types of neurons and glia cells throughout the CNS and PNS and affects many neural functions such as behavior, cognition, and motor control.120In the literature COMT gene enzyme catecholamine-O-methyltransferase that inactivates or catabolizes the neurotransmitters dopamine, norepinephrine (NE), epinephrine, as well as caffeine and estrogens has been well studied in the humans and rodents.34Similarly, single nucleotide polymorphism (SNPs) in the COMT gene have been assessed to understand the development of TMD pain. individual SNPs provide important insights into etiology, while complex conditions such as TMD are likely influenced by combined effects of multiple SNPs operating through specific biological pathways. In OPPERA study, three hundred genes were investigated, implicating six SNPs as risk factors for chronic TMD. One SNP was in the glucocorticoid receptor gene, suggesting a contribution of the hypothalamic-pituitary-adrenal system to chronic TMD. Another SNP was in the serotonin receptor gene, supporting other studies indicating that the gene influences nociceptive and affective pathways. Next was the gene encoding the alpha subunit of the voltage-gated sodium channel Nav1.1, which influences action potentials in sensory nerves. It was associated with nonspecific orofacial symptoms such as jaw stiffness and fatigue. Also, psychological and somatic symptoms were associated with an SNP in the prostaglandin-endoperoxide synthase 1 gene, also known as COX-1, which regulates nociception and inflammatory response has been noticed as well as variation in the gene encoding APP, amyloid beta (A4) precursor protein, which affects synapse formation and neuronal plasticity were found to be associated with psychological stress and negative affectivity. Finally, heat pain temporal summation was associated with an SNP in the multiple PDZ domain protein gene, which influences G protein-coupled receptors involved in nociception and analgesia has been assessed.40 Single nucleotide polymorphisms (SNPs) coding cytokines and transcription factors were genotyped. They observed that TMD cases with and without widespread pain. Researchers observed that TMD cases with and without widespread pain had inhibited transcription activity of the anti-inflammatory cytokine transforming growth factor β1 (TGFβ1). They reported interactions between TGFβ1 and IL-8 SNPs an additional copy of the TGFβ1 rs2241719 minor T allele which was associated with twice the odds of TMD cases with widespread pain among individuals homozygous for the IL-8 rs4073 major A allele, and half the odds of TMD cases with widespread pain among individuals heterozygous for rs4073. These results demonstrate how pro- and anti-inflammatory cytokines contribute to the pathophysiology of TMD and wide-spread pain in genetically susceptible people.40In a study,119 They evaluated the relationship between multisystem dysregulation and the presence of TMD pain, as well as the presence of different numbers of comorbid pain conditions in TMD. In this study twenty markers from sensory, autonomic, inflammatory, and psychological domains were evaluated. The results revealed that first overall dysregulation in multiple system domains , particularly in the sensory  and the psychological  domains, were associated with increased likelihood of being a painful TMD case and second dysregulations in individual system domains were selectively associated with the increased odds of being a TMD case with different levels of comorbid persistent pain conditions. These outcomes indicate that heterogeneous multisystem dysregulations may exist in painful TMD subgroups, and multidimensional physiological and psychological assessments can provide important information regarding pathophysiology, diagnosis, and management of pain in TMD patients.119In a study, Chen et al. reported that multiple physiological and psychological regulatory domains may contribute to the pathophysiology of pain in temporomandibular disorder (TMD) and other bodily pain conditions.120Further, based on the combination of genotypes of these SNP investigators categorize the haplotypes as low, average, and high pain sensitivity . Later, investigators concluded that individuals with the LPS haplotype have higher levels of COMT enzymatic activity, which means decreased probability of developing TMD while APS or HPS haplotypes had an increased relative risk of developing TMD compared to those with “pain-resistant” LPS haplotypes.120 Additionally, in chronic widespread pain conditions, such as fibromyalgia, that often overlap with TMD genome-wide association studies suggest that genetic and epigenetic factors such as SLC64A4, TRPV2, MYT1L, and NRXN3 along with environmental factors play a vital role in the development of chronic pain.However, genetic association studies in TMD concluded that TMD heritability is multifaceted that mostly arises from genes encoding proteins involved in the serotonergic and catecholaminergic systems. The OPPERA study identified associations between TMD-related pain and SNPs in COMT, the serotonin receptor HTR2A, and ERA and observed that TMD-related pain, are significantly more prevalent in women than men.121 Similarly, oxidative stress biomarkers markers 8-hydroxydeoxyguanosine and malondialdehyde (8‐OHdG) have also been analyzed. In a recent observation, higher levels of 8‐OHdG from saliva  or log 8‐OHdG from serum (OR: 2.22, 95%CI: 1.71–2.88, were both related to severity of TMD pain and total antioxidant status have been found to be significantly different in saliva between TMD patients and controls.123There are a few studies that also have analyze the biochemical changes associated with TMD. In the study, a reduced levels of vitamin D were observed in the patients with TMD compared to control.Current review elucidates the potential biomarkers involved in TMD pain. Summary of Potential Biomarkers are depicted in Table It is still needed to be explored whether these potential biomarkers can fulfill considerations of reliability and practicality in terms of replication of studies on heterogenous and large-scale sample size populations. In future, more clinical and experimental biomarkers research is needed on heterogenous models and patients with TMD pain and other comorbid conditions. Machine learning approaches have yielded promising results towards making predictions about states of pain in humans and animals based on neural recordings. Furthermore, discovery of new biomarkers will provide valuable insights in understanding of TMD pain pattern and develop therapeutics methods which can be integrated in the treatment of TMD.


Tightly orchestrated programmed cell death (PCD) signalling events occur during normal neuronal development in a spatially and temporally restricted manner to establish the neural architecture and shaping the CNS. Abnormalities in PCD signalling cascades, such as apoptosis, necroptosis, pyroptosis, ferroptosis, and cell death associated with autophagy as well as in unprogrammed necrosis can be observed in the pathogenesis of various neurological diseases. These cell deaths can be activated in response to various forms of cellular stress (exerted by intracellular or extracellular stimuli) and inflammatory processes. Aberrant activation of PCD pathways is a common feature in neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), Alzheimer’s disease, Parkinson’s disease, and Huntington’s disease, resulting in unwanted loss of neuronal cells and function. Conversely, inactivation of PCD is thought to contribute to the development of brain cancers and to impact their response to therapy. For many neurodegenerative diseases and brain cancers current treatment strategies have only modest effect, engendering the need for investigations into the origins of these diseases. With many diseases of the brain displaying aberrations in PCD pathways, it appears that agents that can either inhibit or induce PCD may be critical components of future therapeutic strategies. The development of such therapies will have to be guided by preclinical studies in animal models that faithfully mimic the human disease. In this review, we briefly describe PCD and unprogrammed cell death processes and the roles they play in contributing to neurodegenerative diseases or tumorigenesis in the brain. We also discuss the interplay between distinct cell death signalling cascades and disease pathogenesis and describe pharmacological agents targeting key players in the cell death signalling pathways that have progressed through to clinical trials. Programmed cell death (PCD) is required for normal development and maintenance of tissue homoeostasis, and the elimination of damaged, infected or obsolete cells in multicellular organisms. Seminal discoveries by Kerr et al. in 1972 identified the hallmark ultrastructural features of cells undergoing programmed suicide, where the term ‘apoptosis’ was coined for this form of PCD . Upon acMcl-1 and one allele of Bclx (Mcl1+/−Bclx+/− mice) causes severe craniofacial abnormalities and early post-natal death, while additional loss of one allele of Bim (Mcl1+/−Bclx+/−Bim+/− mice) prevents these abnormalities completely [Normal development and tissue homoeostasis in multicellular organisms depend on orchestrated PCD signalling events that are tightly regulated. During embryogenesis, the elimination of cells by PCD is necessary for adequate moulding of certain tissues, for example the sculpting of the digits of vertebrate limbs . The cenmpletely .While removal of superfluous neuronal cells is vital for normal brain function, aberrant death of distinct neuronal cell populations is a hallmark of pathology associated with neurodegenerative diseases, such as ALS, Alzheimer’s disease (AD), Parkinson’s disease (PD) and Huntington’s disease (HD) (reviewed in ). ConverThe aetiology of neurodegenerative diseases is multifactorial, being associated with defects in different cellular processes, such as response to oxidative stress, excitotoxicity, mitochondrial dysfunction, protein misfolding (ER stress) and inflammation –19. ConsIn this review, we provide a brief overview of PCD pathways, namely apoptosis, necroptosis, pyroptosis, ferroptosis as well as cell death associated with autophagy and unprogrammed necrosis. We discuss their known and proposed roles in the pathogenesis of neurodegenerative diseases and brain cancer, such as GBM. We also describe already existing and proposed therapeutic strategies to target central regulators of the various PCD pathways for the treatment of neurological diseases.Apoptosis can be triggered by two distinct pathways: the intrinsic  pathway and the death receptor  pathways , 21. TheG93A mouse model [G85R transgenic mice and, importantly, BIM deficiency extended the lifespan of SOD1G93A mutant mice [G93A mice by administration of the broad spectrum caspase inhibitor zVAD-fmk [Amyotrophic lateral sclerosis (ALS) is a progressive adult-onset form of MND caused by the aberrant death of motor neurons in the cerebral cortex and spinal cord. The resulting motor neuron loss leads to muscle atrophy and weakness, muscle twitches, spasticity and typically death from respiratory failure . Variousse model , 32 as wse model . Abnormaant mice . The onszVAD-fmk , overexpzVAD-fmk  or the azVAD-fmk .AD is the leading cause of dementia. Hallmark pathological features of AD include the accumulation of amyloid-β-containing neuritic plaques derived from aggregates produced by sequential cleavages of the amyloid precursor protein (APP), neurofibrillary tangles and dystrophic neurites containing hyperphosphorylated tau . EvidencHTT gene that encodes huntingtin. Observations in mouse models of HD include increased levels of pro-apoptotic BIM and BAX in brain lysates at late stages of the disease [Bim significantly attenuated the accumulation of mHTT (mutant Huntington protein), neuronal death and disease-associated phenotypes [HD is characterised by progressive motor, behavioural and cognitive decline. This is driven by expanded CAG repeats in the  disease , 46. Notenotypes . BAX expenotypes .PRKN, LRRK2, PINK1 and PARK7 [Degeneration of the dopaminergic neurons in the substantia nigra is the cause of motor dysfunction in PD that is characterised by a resting tremor as well as abnormal posture and gait. In PD, the predominant mechanism of neuronal death is thought to be via the intrinsic apoptosis pathway in dopaminergic neurons. Inherited forms of PD are associated with mutations in genes associated with mitochondrial function, such as nd PARK7 . The funnd PARK7 . Caspasend PARK7 . Examinand PARK7  as well nd PARK7 . These fGBM is the most common form of brain cancer in humans with a poor prognosis  that is largely due to its invasiveness. GBM cells are resistant to apoptotic stimuli and it is thought that this contributes to the failure of conventional standard of care treatment in this disease. Human glioblastoma cells were shown to express higher levels of BCL-2 and BCL-XL compared to non-neoplastic glial cells . RNA intNecroptosis is a lytic form of PCD that can drive inflammation. Necroptosis can be induced by the stimulation of TNFR1, TLRs and certain other receptors when the activity of caspase-8 is blocked by pharmacological agents or viral inhibitors . This prG93A mutant mice [(OPA1) gene from death and reduced oxidative stress [In ALS, necroptosis was reported to be dispensable for motor neuron degeneration, based on the observation that the absence of MLKL did not affect disease onset, progression and survival in SOD1ant mice . In contant mice , 63. It ant mice . It remaant mice . Pharmacant mice . Therefoant mice . In a tie stress .exon 1 of a mutated human HTT gene is expressed and driven by the human huntingtin promoter [Evidence of a role of necroptosis in the pathogenesis of HD is limited. One study reported that in the R6/2 transgenic mouse model of HD, in which promoter , treatmepromoter . HoweverStroke constitutes the second leading cause of mortality after ischaemic heart disease. It is caused by insufficient blood flow to the brain, triggering a cascade of pathological responses, including inflammation, ROS production and protein misfolding . In animIn cancer, there are contradictory reports claiming that necroptosis can either promote or inhibit tumour growth , perhapsAutophagy is a highly conserved process for the degradation of macro-molecular structures and even entire organelles that plays critical roles in cellular and tissue homoeostasis . This prSQSTM1, OPTN, TBK1, VCP and C9ORF72, are associated with familial forms of the disease. The accumulation of autophagosomes in the cytoplasm of spinal cord neurons of ALS patients has been reported [G93A mouse model of ALS loss of Atg7 accelerated neuromuscular denervation and the onset of hindlimb tremor at the early-symptomatic stage of the disease. However, at late stages of disease, autophagy had an adverse role, where loss of Atg7 slowed disease progression and increased the lifespan of the mice [A hallmark of neurodegenerative diseases includes the accumulation of proteinaceous aggregates and ubiquitinated inclusion bodies and they are thought to be involved in the aetiology of these diseases. Aberrant autophagy is a feature of several neurological diseases. In ALS, mutations in autophagy-related genes, including reported , as wellreported . In the the mice .The accumulation of autophagosomes in neurons is a conspicuous feature of AD in both animal models and patients with observations of increased Aβ generation and accumulation in lysosomes in cells with defects in autophagy. This suggests that the turnover of Aβ is regulated in part by autophagy . MicroarATG genes were shown to be aberrant in PD [Defects in autophagy are associated with several molecular mechanisms underpinning PD, and several nt in PD . Accordint in PD .It is a similar scenario in HD, where accumulation of the huntingtin protein (HTT) is associated with attenuated autophagy . NotablyThe role of autophagy in GBM is controversial, with some reports claiming that it suppresses tumour growth whereas others stated that it promotes tumour growth. In U343 glioma cells, autophagy was shown to trigger cell senescence  and thisFirst coined in 2012 , ferroptMany small molecule inhibitors are redox active and exhibit significant potential to directly inhibit lipid peroxidation . This isGpx4 knockout mice [Specific evidence for a role of ferroptosis in a disease setting is difficult to establish. Almost all neurodegenerative diseases appear to exhibit lipid peroxidation. Likewise, dysregulated iron homoeostasis and diminished glutathione are also common features of neurodegeneration. Perhaps the best evidence comes from the protection afforded by ferroptosis inhibitors in animal models of disease and ultimately in human clinical trials. The canonical RTA ferroptosis inhibitors liproxstatin-1 and ferrostatin-1 have been reported to exhibit efficacy in mouse models of stroke , PD 111111 and Hout mice , 114, whout mice .Several ferroptosis inhibitors have been assessed in the clinic. In a phase II clinic trial for AD, the iron chelator deferoxamine significantly reduced the rate of cognitive decline in patients . SurprisMore recently, therapeutic strategies targeting iron have focused on the iron chelator deferiprone. Similar to deferoxamine, deferiprone inhibits ferroptosis in vitro, and exhibits efficacy in mouse models of AD, PD and ALS –121. In II(atsm) strongly inhibits ferroptosis induced by RSL3 or erastin in neural cells in vitro and in a cell-free lipid peroxidation system [II(atsm) has been extensively investigated  in preclinical animal models and exhibits efficacy in multiple models of ALS, PD and stroke (for review see [Cun system . CuII(atview see ). Phase view see . A phaseFerroptosis-related gene expression is associated with diagnostic and prognostic factors in glioma . Many anPyroptosis is an inflammatory form of PCD involving activation of caspase-1 by inflammasomes. Caspase-1 proteolytically processes pro-IL-1β and pro-IL-18 into the mature inflammatory cytokines IL-1β and IL-18, respectively. Gasdermin D (GSDMD) is the critical executioner of pyroptosis . CaspaseEvidence for pyroptosis (accompanied by inflammasome activation and elevated IL-1β and IL-18) has been reported for many neurodegenerative diseases, including AD, PD, ALS, HD, multiple sclerosis, stroke and traumatic brain injury . Inflamm2O2), ischaemia-reperfusion and calcium overload [2+ concentration and generation of reactive oxygen species culminating in events that result in irreversible cell injury. However, unlike necroptosis, necrosis lacks a defined core cellular signalling machinery but Ninj1 has recently been identified as being critical for the rupture of the plasma membrane [Necrosis is classically considered an unprogrammed and unregulated cell death process that is characterised by cell swelling, loss of membrane integrity, ‘spillage’ of intracellular contents (DAMPs and PAMPs) into the extracellular environment and dissipation of ion gradients, overall triggering an inflammatory response. Necrosis can occur due to overwhelming stimuli from outside the cell, such as hypoxia, freezing or burning, certain pathogens, physico-chemical stresses  mimicking neurological diseases has led to an understanding of key regulators of the different cell death signalling pathways and their relevance in disease pathogenesis. Even though aberrant apoptotic cell death and expression of key pro-apoptotic proteins are associated with neurodegenerative diseases, targeting apoptosis in vivo has so far proved disappointing. The clinical potential of Minocycline, a second-generation tetracycline was tested in various preclinical mouse models of neurodegenerative diseases, such as ones for ALS, PD and HD. Minocycline blocks the release of cytochrome c from mitochondria, inhibiting this step in apoptosis, and was reported to upregulate the expression of anti-apoptotic BCL-2, also exhibiting anti-inflammatory and antioxidant effects, displaying effective neuroprotective outcomes in preclinical mouse models . HoweverPharmacological inhibition or genetic ablation of RIPK1 has produced neuroprotective outcomes in preclinical models of AD, PD and HD. The use of the blood brain barrier-penetrant RIPK1 inhibitor DNL747 was tested in a clinical trial for AD and ALS, even though necroptosis was reported to be dispensable for the latter. DNL747 progressed through phase I trials. However, the trial was then halted in favour of its successor compound, DNL788, which is anticipated to be superior in achieving neuroprotective outcomes . The effBcl2l12, a protein containing a BH2 domain (but none of the other BCL-2 family homology (BH) regions) was reported to drive the development of GBM by interacting with certain members of the BCL-2 protein family and inhibiting the activation of caspases-3 and -7, thereby inhibiting mitochondrial-induced apoptosis . An earlRegulators and effectors of the different cell death pathways remain attractive therapeutic targets that may form the basis for translational work that will hopefully lead to improvements for patients with these diseases. Given that the aetiology of neurological diseases is complex, where multiple cell death mechanisms often in conjunction with other cellular processes drive pathology, it appears likely that effective therapies will comprise inhibitors of more than one cell death programme plus inhibitors of additional cellular processes. Table Many diseases of the brain are associated with defects in one or several processes of PCD: either aberrant killing of cells that should survive in neurodegenerative disorders or aberrant survival of cells that should die during the development and therapy of brain cancers. In most neurodegenerative diseases it has not yet been unequivocally defined whether these defects in cell death are a true cause or at least critical contributor to disease or simply a consequence of some insult to the tissue where even effective blockade of this cell death would not offer improved therapy. In the case of brain cancer, as with all types of cancer, effective delivery of potent inducers of any type of cell death would be expected to cause tumour shrinkage, of course with the proviso that it will be safe, i.e. tolerable to vital tissues. We contend that much additional research, both basic work in animal models and studies with patient material, is needed to garner a more detailed understanding of the roles of the different processes of cell death in diseases of the brain, so that this knowledge can be harnessed to develop truly transformative advances in their treatment.Neurodegenerative diseases are associated with aberrant neuronal cell death, but the processes leading to such cell death and the mode of cell death still remain unclear.Glioblastoma cells are highly resistant to apoptosis; therefore, activation of alternate mechanisms of PCD may represent promising avenues to explore for therapy of this cancer.With the brain’s complex cellular and architectual diversity, it is not unreasonable to predict that many forms of cell death may be occurring simultaneously in disease states. Indeed, evidence indicates this is likely the case, with various inhibitors targeting many forms of cell death having beneficial impact in the same disease model.Given there are perturbations in more than one PCD signalling pathway and also unprogrammed necrosis in disease pathogenesis, effective therapies will need to comprise inhibitors of more than one type of cell death.Is a defect in a PCD signalling pathway a direct cause or a critical contributor to disease or simply a consequence of some insult to the tissue where even effective blockade of cell death would not offer improved outcome?Programmed pathways of lytic cell death are inherently more immunogenic than apoptosis. Will inhibition of lytic PCD pathways be more effective in the treatment of neurodegenerative diseases than blocking apoptosis because the former induces a pro-inflammatory state? Are neurons more susceptible to death in an inflammatory state?The ferroptosis PCD pathway is not immune-silent. What impact would this have on surrounding healthy tissues when trying to exploit inducers of ferroptosis for the treatment of brain cancers?


Mitochondria are essential cellular organelles, involved in controlling energy production, cell metabolism, cell growth, and cell death. Since cellular functions are de-regulated in cancer, it is not surprising that mitochondria dysfunctions have been observed in this disease. One key aspect in controlling tumor formation and progression is the interaction between the cancer cells and their surrounding environment, known as the tumor microenvironment. The extracellular matrix is an abundant component of the tumor microenvironment and it has been shown to affect tumor initiation and progression. Here, we will explore how the interaction between cancer cells and the extracellular matrix impinges on mitochondria function during cancer progression.The tumor microenvironment, in particular the extracellular matrix (ECM), plays a pivotal role in controlling tumor initiation and progression. In particular, the interaction between cancer cells and the ECM promotes cancer cell growth and invasion, leading to the formation of distant metastasis. Alterations in cancer cell metabolism is a key hallmark of cancer, which is often associated with alterations in mitochondrial dynamics. Recent research highlighted that, changes in mitochondrial dynamics are associated with cancer migration and metastasis—these has been extensively reviewed elsewhere. However, less is known about the interplay between the extracellular matrix and mitochondria functions. In this review, we will highlight how ECM remodeling associated with tumorigenesis contribute to the regulation of mitochondrial function, ultimately promoting cancer cell metabolic plasticity, able to fuel cancer invasion and metastasis. Cancer cells can change their metabolism according to their progression and their needs which can be provided by the interaction with the surrounding microenvironment . Even thMitochondria are highly dynamic organelles where their structure, function, and cellular distribution can reflect the metabolic changes. Their dynamics are usually represented by two processes, fission and fusion. Fission is a process where mitochondria are divided into multiple structures to generate new ones, thereby facilitating mitochondria relocation to cellular regions of high energy demand. Fission is mainly regulated by Dynamin-related protein (Drp1). Fusion is a process where numerus mitochondria are fused including damaged ones and is regulated by mitofusins (Mfns) and optic atrophy-1 (OPA1). When fusion reduces, the fragmentation of mitochondria into small, spherical mitochondria results in impaired respiratory function, while stimulation of fusion results in the formation of a highly connected mitochondria network ,3,4.The dysregulation in mitochondria fission and fusion processes affects both mitochondria structure and function, and it has been linked with human disease. Indeed, mutations in the regulators of mitochondria dynamics have been linked with several neurodevelopmental and neurodegenerative diseases. Mitophagy, which is a type of autophagy-promoting damaged mitochondria elimination, plays a role in the quality control and morphology of mitochondria ,6. In caTumors are surrounded by multiple components which form the tumor microenvironment (TME). The TME consists of different cell types such as endothelial cells, immune cells, and fibroblasts, in addition to the extracellular matrix (ECM) which provides mechanical and biochemical support to the resident cells . ECM plaDue to the high tumor growth rate and the limited blood supply, the tumor microenvironment has been found to be hypoxic and deprived of nutrients . TherefoAccumulating evidence suggest that there is a link between the ECM and mitochondria and that mitochondria can sense any changes in the TME including changes in the ECM such as its composition and stiffness. In this review, we will discuss the interplay between the ECM and mitochondria and its role in controlling mitochondria morphology and dynamics.ECM forms most of the TME in many tumors and it comprises hundreds of proteins—altogether defined as the “matrisome” manly secreted by cancer cells themselves and by the cancer-associated fibroblasts (CAFs) . The matCancer cells interact with the ECM via cell membrane receptors such as integrins. Integrins are heterodimers which consist of two subunits, α and β. They are activated by binding to their ECM ligands which then trigger multiple signaling pathways that promote cell proliferation and survival . IntegriMore recently, it has been established that adhesion complexes also play an important role in controlling nutrient sensing. In particular, the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) has been shown to be activated at paxillin-containing focal adhesion, through the upregulation of localized amino acid uptake. In addition, fibronectin-bound integrin has been shown to be internalized specifically from fibrillar adhesion in a tensin-dependent manner. This internalization pathway supports lysosomal clustering and mTOR activation in ovarian cancer cells. Finally, the energy sensor AMPK has been shown to prevent tensin-dependent integrin activation in fibroblasts ,18,19. IPaxillin (PAX) is a focal adhesion protein that interacts with FAK, promoting FAK-Src binding and the downstream signaling pathways to link integrins to the actin cytoskeleton . It has PINCH1 and 2 are focal adhesion proteins expressed in mammalian cells . They inIn cultured endothelial cells, αvβ3 integrin has also been shown to support mitochondria function, via the activation of a FAK-signal transducer and activator of transcription 3 (STAT3) signaling pathway induced by vitronectin binding . STAT3 h2−), and the hydrogen peroxide (H2O2) . T. T39]. TMetastasis is a multi-step process where cancer cells migrate from the primary site, invade into the ECM, enter the blood vessels, and reach a secondary site . Cancer The dysregulation in mitochondrial dynamics has been linked to cancer invasion. Indeed, mitochondria fission and the expression of Drp1 have been found to be enhanced in highly invasive breast cancer cells. The fission event is necessary for the redistribution and the movement of mitochondria to the lamellipodia region of the cells, where the energy demand is high. The formation of lamellipodia was opposed by inhibiting DRP1, as well as cell migration and the invasion after performing matrigel invasion and migration assays . These rMitochondria trafficking to the leading edge of the cells has also been found to be a way for cancer cells to bypass PI3K inhibitors and this was accompanied by a growth-factor receptor-mediated Akt and mTOR re-phosphorylation. The accumulation of energetically active elongated mitochondria at the leading edge promoted membrane dynamics, focal adhesion turnover, and cell migration. Interestingly, the inhibition of the mitochondria fusion effect on mitofusion1 opposed mitochondria trafficking and cells invasion induced by PI3K inhibitors . This suThe role of mitochondria in cancer cells migration and metastasis has been extensively reviewed elsewhere ,46. HoweThe ECM architecture can also affect the ATP:ADP ratio inside the cells. By increasing collagen I density from 2D to 3D matrices and by changing it from aligned to random fibers, the ATP:ADP ratio increased inside the cells B 48]. T. T48]. TThe stiffness of ECM has been found to play a role in mitochondria dynamics by two different mechanisms; soft collagen I has been shown to trigger mitochondria fission in human mesenchymal stem cells (MSCs) in a kindlin-2 dependent process, while stiff ECM can trigger mitochondria fusion and an inhibition of DRP1 in a PINCH1-dependent manner C 50]. M. M50]. MDuring pancreatic cancer progression, ECM deposition is dramatically increased, and ECM stiffness in this context has been shown to affect mitochondria dynamics. In ductal adenocarcinoma cell lines, mitochondria become more elongated in stiff ECM, and they accumulate in invasive protrusions to fulfil the ATP demand associated with cytoskeletal remodeling and cell migration . ATP recSeveral anti-cancer therapies target cell proliferation and growth, however metabolic reprograming and plasticity of cancer cells enable them to restore their ability to grow and survive. Thus, understanding the tumor metabolic adaptabilities will be beneficial to finding new targets which could improve the existing therapies and avoid the potential resistance. Interestingly, ECM stiffness has been found to play a role in cancer cells resistance to chemotherapy. A recent study by Romani et al. showed that, being in a soft microenvironment, metastatic breast cancer cells can overcome oxidative stress and ROS-dependent chemotherapy drugs via the transcription factor NRF2 (nuclear factor erthroid-2-related factor 2). Soft ECM enhances mitochondria fission due to increased expression of DRP1 and mitochondria elongation factor 1 and 2 (MIEF1/2). In this case, mitochondria fission increases both the production of mtROS and the activity of NRF2 antioxidant transcriptional response, therefore cells increase their tolerance to oxidative stress . It willOne of the main aspects that the cells need to control is the balance between survival and apoptosis. ECM detachment results in cell apoptosis through a process called anoikis . MitochoIt has been found that ECM-detached cells can activate the receptor-interacting protein kinase 1 (RIPK1), which in turn induces mitophagy, mediated by the mitochondrial phosphatase phosphoglycerate mutase 5 (PGAM5), leading to ROS induction and a non-apoptotic cell death B 57]. H. H57]. H2+ alterations. However, what is the role of Ca2+ in this process and how it is regulated still need to be investigated. By adding exogenous fibronectin, the apoptotic process was reversed . H. H65]. HAltered metabolism has been identified as one of the hallmarks of cancer  where ca


Alu domains, highlighting their role inmodification of gene structure and gene expression in addition to theirpolymorphic nature being a genetic modifier of disease risk and progression.Although the literature focuses on their role in disease, it illustrates theirpotential to be major contributors to normal physiological function. To date,these elements have been under-reported in genomic analysis due to thedifficulties in their characterization with short read DNA sequencing methods.However, recent advances in long read sequencing methods should resolve theseproblems allowing for a greater understanding of their contribution to a host ofgenomic and functional mechanisms underlying physiology and disease.Understanding the mechanisms regulating tissue specific and stimulus inducibleregulation is at the heart of understanding human biology and how thistranslates to wellbeing, the ageing process, and disease progression.Polymorphic DNA variation is superimposed as an extra layer of complexity insuch processes which underpin our individuality and are the focus ofpersonalized medicine. This review focuses on the role and action of repetitiveDNA, specifically variable number tandem repeats andSINE-VNTR- Interpretation of the functional consequences of human genome variation is anessential part of modern biomedical research. This review describes the impact ofrepetitive DNA on experimental biology by allowing insight into the role ofnon-coding DNA in regulation of gene structure and function. The genetic variationinherent in repetitive DNA is becoming an increasing important parameter inpersonalized medicine. Emerging long read DNA sequencing technologies should aid theimproved characterization of these elements in the human genome which will integratethat knowledge into clinical practice and improve the precision of clinicaldiagnoses and decision-making. This review should prove a valuable resource to thefield to capture the distinct mechanism utilized by repetitive DNA in genefunction.E. coli cloning strategies used in the construction of thelibrary of DNA sequences incorporated into the reference genome, and thedifficulties in interpreting and reassembling sequence from such short readsequences in areas of repetitive DNA. The advent of long-read sequencingtechnologies will help overcome the latter problem, and technologies such as OxfordNanopore or Pacific Biosciences single-molecule real-time sequencing (PacBio) caneasily read 10 kb or more sequence; furthermore, base calling accuracy has improved(error rate said to be <1% for PacBio and <5% for Oxford Nanopore) andanalytical pipelines to call structural variation have been developed. This review will focus on one class of repetitive DNA, namely variable numbertandem repeats (VNTRs), highlighting their functional roles in genome regulation andas biomarkers of disease focusing on neurological diseases and disordersNinety-eight percent of the human genome does not code for proteins, but contains avariety of regulatory elements including those to direct (a) tissue specific andstimulus inducible gene expression, (b) differential mRNA splicing to generatedistinct protein profiles or turnover, and (c) functional non-coding RNAs. Theproperties of these regulatory components like the exons themselves can be modifiedby genetic polymorphism or mutations. To date, genome analyses have favored shortread next generation DNA sequencing and genome-wide association studies (GWAS). Thelatter has focused on analyzing variation in single nucleotide polymorphisms (SNPs)and has identified genomic variation in strong association with phenotype both inexons and non-coding regions. Such SNPs in exons are often easy to interpretmechanistically as many will change a key amino acid providing an explanation forthe phenotypic association. However, the functional significance of the vastmajority of SNPs in non-coding DNA is difficult to determine; furthermore, a majorshortfall of this approach is that it has neglected the role and function of othersources of genetic variation in the genome such as that represented by repetitiveDNA. This is in part due to the inherent instability of human repetitive DNA in24 In modern humans, TR DNA hasbeen associated both with traits such as aggression and addictive behaviors,reviewed by literature58 and implicated in the increasedsusceptibility to, and risk of developing, a wide variety of neurological diseasessuch as motor neuron disease911 andAlzheimer’s disease 13 mental health conditions such as bipolar disorder, depression,and schizophrenia.15 To date, 1584 such repeats have been found to be human specificand it has been proposed that they contribute to human-specific traits. When the repeat number is variable, i.e. polymorphic, they become VNTRs, aclass which accounts for approximately 3% of the human genome17; such repeatelements can vary in copy number and additionally demonstrate single nucleotidevariation within the repeats or even short insertions or deletions. There areseveral mechanisms proposed, by which this variation could develop, including errorsin DNA replication such as slipped-strand mispairing which results in themisalignment of DNA strands and thus expansion or contraction of the copy number ofthe DNA motifs, homologous recombination, and duplications. Recently it has been shown 55% ofVNTRs map to the terminal 5Mbp of human chromosomes,20 and regions that havepreviously been found to demonstrate increased double-strand breaks during earlystages of meiosis22 and male meiotic recombination. VNTRs may be located withinexons, introns, or intergenic spaces and this aforementioned group of VNTRs appearsto favor intronic locations. A second pathway contributing to the derivation of VNTRs is viaretrotransposition, particularly utilizing the compositeSINE-VNTR-Alu (SVA) element, and these elements tend to showbias against incorporation in to genic regions, and have a high GC content. Unsurprisingly, VNTRs located within exons are the smallest group. Originallythe main focus of research was on short tandem repeats (STR) (or micro-satellites)as it became apparent that expansions of these can occur in somatic cells, and thisphenomenon is implicated in diseases such as fragile X-associated tremor/ataxiasyndrome, myotonic dystrophy, Huntington’s disease, spinocerebellar ataxia, andamyotrophic lateral sclerosis (ALS).2333 This approach originallyidentified a number of diseases associated with triplet expansions, i.e. Fragile Xsyndrome in 1991  and Huntington’s disease in 1993; however, expansions of longer repeat sequences have since been found. For thelatter, examples include: pentanucleotide expansions in the genesSAMD12 and RFC1 found in benign adult familialmyoclonal epilepsy type1 and ataxia syndromes, respectively,36 ahexanucleotide repeat expansion in the C9ORF72 gene associated withALS,33 and a 12-nucleotide expansion in the CSTB geneassociated with progressive myoclonic epilepsy of the Unverricht-Lundberg type orEPM1   DNA imparts a huge source of variation to the genome, and TRs haveevolved throughout evolution and contribute to genetic diversity. Although they arefound in many eukaryote species, present day interest has focused on their role inhominid evolution and their contribution to the development of human-specific traitsin the evolution of modern humans and in particular neural function.rEPM1  . Todatein vivo and in vitro. Perhaps one of the earliestand most striking illustrations of allele-specific activity, was the invivo demonstration of the ability of the human-specificSLC6A4 intron 2 VNTR to direct differential reporter geneexpression in the midbrain of mouse embryos equivalent to the area where the mouseserotonin transporter is initially expressed in the developing brain.50 Associationstudies have established a link between repeat copy number in this VNTR as a riskfactor in various neurological diseases or disorders; however, establishing thefunctional significance of such VNTR polymorphism in situ is moreproblematic. Some of this difficulty may be attributed to non-coding VNTRs beingregulatory domains that are only functional in specific tissues, developmentalstages, or in response to specific cellular challenges. Recent examples of VNTRsfound to have regulatory properties include the finding that the longer risk allelesof the intronic VNTR in ABCA7 favored use of a cryptic splice siteresulting in exon skipping  and that the number of VNTR repeats in the promoter region ofTRIB3 correlated with mRNA levels in various tissues.An increasing number of VNTRs have been identified which support differential geneexpression both MIR137 locus works together with the SNPrs2660304 to drive differential promoter activity in vitro,thereafter it was shown using haplotype analysis at the MIR137locus that rs2660304 was a proxy SNP for the schizophrenia GWAS SNP rs1625579. Furthermore, interactions between multiple VNTRs to regulate gene expressionare possible. In vitro analysis has demonstrated that distinctVNTRs within a gene locus can act either independently or synergistically toregulate transcription. For example, the serotonin transporter(SLC6A4) gene which has VNTRs located both in the linkedpolymorphic region of the 5ʹ promoter and intron 2 which have been shown to act combinatorically, a further example is the MAOA gene which in addition to thewell-characterized µVNTR has a second distal (d)VNTR located approximately 500 bp upstream of the µVNTR. The complexity this produces was illustrated by the recent meta-analysis ofMAOA by Tunbridge et al. where it was foundthat the high and low activity µ alleles were associated with enzyme activity in theblood but did not affect MAOA mRNA abundance; concurrently, Manca et al. demonstrated that the distal VNTRregulated mRNA levels of the canonical isoform of MAOA in a cell line model. Moreover, specific regulatory effects on MAOA isoforms and an additive effectof the two VNTRs at this locus were highlighted in additional work. A further layer of intricacy was uncovered by the finding that VNTR specific responses to a stimulus also existed. Thus, it is important to consider the overall haplotype when analyzing generegulation rather than assuming a single regulatory variation is solely associatedwith a specific phenotype.53If VNTRs are involved in transcriptional or post transcriptional gene regulation thenthey will act in consort with other gene regulatory domains to direct tissuespecific and stimulus inducible gene expression. For example, it has been shown thata VNTR located at the 5557 Morespecifically in the context of neurological disorders, the previously mentionedSLC6A4 intron 2 VNTR has been assessed via reporter gene assaysin rat prefrontal cortical cells, where it was found that the different copy numbervariants induced reduced but differential reporter gene expression in response toCCCTC-binding factor (CTCF). Furthermore, it has been shown that both SLC6A4 VNTRs canbind multiple transcription factors, including y-box binding protein (YB1), CTCF andmethyl-CpG binding protein (MeCP2), inducing allele-specific expression profiles inresponse to cocaine. A further example is the VNTR identified in the 3ʹ UTR of the human dopaminetransporter (SLC6A3) gene, which has been associated withattention-deficit hyperactivity disorder.41 Previously it had been shownusing reporter gene assays in the SH-SY5Y cell line that the SLC6A3VNTR induced significant repression of luciferase expression in response to the HESR(HEY) family of transcription factors, specifically HESR1 and HESR2.In addition to the more conventional mechanism of provision of transcription factor(TF) binding domains by which VNTRs can contribute to the regulation oftranscription, there are several examples of them encoding miRNAs and affectingepigenetic parameters . In geneWDR7 and DNAJC5 which harbors the human-specific miR-941 withinan intronic VNTR. This latter example displays unusual features for a recently emergedhuman-specific miRNA, namely high levels of expression, particularly of note withregard to this review, in the cerebellum and prefrontal cortex and the copy numberdemonstrates a high level of variability. Human-specific regulation by this miRNAhas been shown in the brain, targets included the host gene DNAJC5whose protein has amongst other functions a role in neurotransmitter release, preventing neurodegeneration  and is associated with adult-onset neuronal ceroid lipofuscinosis. Furthermore, this poses the question does the copy number of the VNTR leadsto distinct levels of the miRNA directly, or is the mechanism indirect, achieved bymodification of the processing of the internal miRNA? The putative miRNA encodedwithin WDR7  has been detected in cytoplasmic aggregates or speckles when experimentallyover-expressed. Such RNA foci, albeit in these cases nuclear, are recognized asimportant features in a number of RNA gain of function disease models such as thatfor myotonic dystrophy  and C9ORF72-associated ALS.64More recently, it has been discovered that some VNTRs have the potential to encodefor miRNAs. This phenomenon has been recently described for VNTRs within the geneset al. regarding the response of the SLC6A4serotonin transporter gene to stimulation by cocaine—differential effects ontranscription factor binding were seen dependant on which VNTR allele was beingexamined and correlated with MeCP2 binding. One clear mechanism is methylation of the VNTR itself, this requires the VNTRto contain CpG targets. Simplistically, the more CpG in the VNTR, the greateropportunity for methylation changes to alter VNTR function. This is perhaps obviousin short repeats of CG, but can also occur in expansion repeats such as the hexamerCCCCGG tandem repeat found inC9ORF72.66 Changes in the status of CpGmethylation have long been noted to affect the stability of repetitive elementscontaining such sequences; furthermore, methylation status has been purported toenable DNA to adopt alternative non-B DNA structural forms such as Z-DNA. Consideration of the methylation status of the VNTR may help explain some ofthe conflicting reports of the association of repetitive elements with traits anddisease. A prime example of this is the MAOA gene reviewed byZiegler and Domschke, and this highlighted that the µVNTR lies in an area thatexhibits differential methylation; furthermore, the level of methylation ispostulated to contribute to the different disease profiles that have been attributedto the MAOA gene, and the location of MAOA on theX chromosome could also contribute to the gender differences seen in the various analyses. This observation was expanded by Manca et al., whodemonstrated in a cell line model that there was allele-specific gene expression andtranscription factor binding with corresponding allele-specific epigenetic marks inthe region encompassing the µVNTR; furthermore, exposure to the mood stabilizersodium valproate resulted in an allele-specific response in transcription factorbinding and epigenetic marks.Epigenetic modification of a VNTR could have significant consequences for allVNTR-directed regulatory mechanisms in both the immediate response to cellularsignalling and the medium- and long-term properties of the VNTR to modulate genefunction. This was illustrated by the analysis undertaken by VasiliouCCCTCC and CCCCGG and variants thereof. The classical model for G4structure is the trimeric repeat of CCCTCCCC found in the MYC gene promoter whichhas been associated with promoter usage for MYC expression.70 Although thisexample is not a VNTR, it has laid the basis for the putative action of such repeatsfound in VNTRs in mechanisms involved in disease progression or initiation asexemplified by the CCCCGGexpansion repeat in the C9ORF72 gene associated with ALS. Such repeats are postulated to form a genomic structure that allows fortorsional strain on one side and opening of the genome on the other thus allowingfor a range of distinct structural parameters to be imposed at local regions of thegenome. A number of transcription factors, both double stranded DNA binding factorssuch as CTCF and Sp1, and single stranded nucleic acid binding factors, such ashnRNPK and YB1, could then bind to such VNTRs to determine localized genomicstructure.The final mechanism to be highlighted is modification of genome structure and inparticular formation of G-quadruplexes (G4). These structures can be generated byrepetition of C-rich hexameric sequences such as Alu (SVA) element, a non-long terminal repeatretrotransposon. Although the total number of SVAs in the human reference genome ismodest (approx. 2700), SVAs are hominid-specific elements with almost 50% of thosecharacterized in the reference genome found to be human specific and have beenpostulated to correlate with the development of hominid lineage-specific traits. They are composite regulatory DNA domains containing multiple regulatorycomponents  which may share related primarysequence but are clearly distinct from one another. The VNTR may also contain morethan one specific repeat motif. SVAs also have the potential to form the structuralcomponents outlined above, namely G-quadruplexes, and this ability is embedded inthe CT hexamer and the central VNTR region where the CpG content can approach60%.VNTRs are often considered as standalone elements in the genome but as discussedearlier they are also key domains of larger functional components such as thecompound SINE-VNTR-mponents  thataffLRIG2 gene, (2) associated with differential RNA expression and disease severity inParkinson’s disease , and (3)causative of disease as seen in X chromosome-linked dystonia Parkinsonism.The association of SVAs with disease may not only be a modifying parameter but canalso be causative of the disease. This supports the main drive of our hypothesisthat VNTRs are not only biomarkers of disease but can also be mechanisticallyinvolved in progression of the disease. This is demonstrated by the observation thatsome SVAs can be polymorphic for their presence or absence in the genome, thus forspecific SVAs this means that they can be (1) associated with methylationdifferences at adjacent promoters as exemplified by the NOTCH2NLC locus which can have as many as 517repeats in affected individuals with neuronal intranuclear inclusion disease-related disorders and facilitated epigenomic profiling of transposable elements.This review highlights the emerging field analyzing the role and importance of VNTRsin regulation of genome function and regulation of the cell transcriptome. Theseelements have the potential to function in many different ways and have beenpostulated to contribute to human evolution. It is apparent, however, that eachindividual element should not be considered alone, rather it is their concertedaction that is important, and this provides a layer of intricacy to regulationallowing an exquisite and specific response to a stimulus. In the recent past, VNTRshave also been found to be linked with many neurological conditions and disordersand their activity is expected to explain some of the missing heritability in suchdisorders. To date, analysis of VNTRs in association studies has often relied onlabor intensive techniques such as polymerase chain reaction (PCR), which itself canbe technically challenging in these domains due to their repetitive nature and insome instances, high GC content. However, with the advent and increased availabilityof long-read sequencing methods, these problems will in part be overcome and allow amore rapid and robust analysis thus aiding the identification of a host of genomicand functional mechanisms underlying our physiology. For example, long-readsequencing has recently enabled the identification of a short tandem repeat (GGC)expansion in the