Diseases introduction

Knowing the functional role of GJs in conditions such as brain ischemia, neurodegenerative diseases, epilepsy, heart arrhythmia, cataracts, and tumors could allow experts to determine and understand the real molecular mechanism of GJ proteins network during disease processes, a practical separation of diseases based on the type of synapse involved, to simplify their study. Consequently, it could also help design treatments to successfully eliminate these conditions. Connexins and pannexins are key proteins to the bone and skeletal muscle development, maintenance, and regeneration. Research has found that connexin channels are present in the bone among osteoblasts, osteoclasts, and osteocytes. Similarly, connexins are important for bone growth and adaptation¹⁰.

The permeability of GJ channels is increased by multiple factors, including low pH levels, high cytosolic free calcium levels, and voltage gradient across the GJ. In vertebrate species, embryonic tissues are easily dissociated by treating them with low concentrations of a proteolytic enzyme, such as trypsin, along with a divalent-cation chelator, such as ethylenediaminetetraacetic acid, to disrupt the protein-protein interactions that hold the cells together.

The connection between cancer development and GJs was first reported by Loewenstein and Kanno when they studied liver cancer using electrophysiology techniques in 1966¹¹. Twenty-four years later, it was already established that GJ mutations were linked to multiple human genetic diseases¹². Nowadays, connexins are known to be related to the development of conditions such as hearing loss, skin disorders, congenital cataracts and other vision disorders, heart arrhythmia, demyelinating diseases, and oculodentodigital dysplasia¹³.

Although the exact percentage of electrical, chemical, or combined synapse participation is currently unknown, a simplification would help unravel the workings of opening neuronal communication channels during different conditions and would allow progress in these areas. At the molecular level, cellular communication plays a decisive role in the development or treatment of these diseases, and of course, the channels through which these signal molecules travel (Fig 3).

Figure 3 A: internal view of Hirudo medicinalis ganglion through epifluorescence using Lucifer yellow injection, neuropil is illuminated, B: internal view of H. medicinalis ganglion through epifluorescence using Neurobiotin, entire ganglion is illuminated, C: superposition of previous images, showing neuropil location in ganglion, D: ventral view of H. medicinalis ganglion - upper side corresponds to the organism's anterior connective tissue, E: control ganglion, red: TMR-dextran, green: Neurobiotin, F: visualization of the structure of a touch-sensitive neuron (T neuron) in ganglion using Neurobiotin. 

Epilepsy

Leading causes of epilepsy include infectious diseases and abnormal brain development. Epilepsy is a neurological disorder that develops as a result of abnormal brain function, neural communication anomalies, or imbalanced neurotransmitters - which are responsible for neurotransmission (i.e., communication between neurons). Hence, epilepsy occurs when neurons occasionally send abnormal signals in the brain²⁰.

After a head injury, brain stroke, or any other accident, the brain might try to self-repair; however, this process can accidentally lead to abnormal brain connections, and thus epilepsy. During the epileptic crisis, many neurons send signals at the same time - as many as 500 times/s. This event causes changes in a person's behavior, movements, feelings, and levels of consciousness. Nowadays, epilepsy is referred to as a spectrum of disorders with a myriad of causes, severities, and effects. Some people may experience convulsions or lose consciousness, whereas others may simply stop what they are doing, have a short lapse of awareness, and stare into space for a moment without knowing what is happening around them²⁰.

In general, a person is considered to have epilepsy after two unprovoked seizures; a "seizure" is a paroxysmal alteration of neurologic function caused by the excessive, hypersynchronous discharge of neurons in the brain, separated by at least 24 h. Provoked seizures are caused by known precipitating factors, such as high fever, nervous system infections, acute traumatic brain injury, or fluctuations in blood sugar, or electrolyte levels. To date, nearly 2.3 million adults and 450,000 children and adolescents in the US suffer from epilepsy. In Mexico, there are about 2 million people with epilepsy, according to the Ministry of Health. Anyone can have epilepsy, men and women, regardless of their race, ethnicity, or age.

There are also severe cases of epilepsy, such as Dravet syndrome. This is a rare but lifelong dysfunction of the brain characterized by medically refractory seizures and causing serious learning disabilities. Current medications can control epilepsy in 60% of sufferers, whereas the remaining 40% experience what is commonly known as drug-resistant epilepsy. Nowadays, treatment options for epilepsy include about 20 different types of drugs, special diets, and surgical techniques, and medical specialty like neurosurgery. Common tests for epilepsy diagnosis include imaging and monitoring techniques (e.g., electroencephalography and magnetoencephalography), patient medical history, blood tests, behavioral tests, and neurological exams. Cx43 is related to cryptogenic epilepsy that is defined as a group of focal or generalized epilepsy, which are believed to be symptomatic of a histopathological or cellular occult alteration, but not of a genetic nature²¹.

Research has found that Cx43 in GJs²¹ and hemichannels²² adversely affects astrocyte function, thereby causing epileptic seizures, which in turn induce Cx43 expression²³. On the other hand, changes in Connexin 36 (Cx36) expression cause cell death²⁴ and neural communication anomalies, thus resulting in epilepsy²⁵. Furthermore, GJs in astrocytes play a crucial role in ionic regulation; particularly, they can change the concentration of potassium ions. Changes in the strength of GJ coupling can cause neuronal hyperexcitability, and consequently, spontaneous epileptic activity²⁶, which ultimately leads to astrocyte uncoupling. Finally, there is evidence that pannexin 1 (Panx1) contributes to the maintenance of epileptic seizures by releasing ATP. The absence of Panx1 in astrocytes potentiates seizure manifestation due to low levels of adenosine kinase, whereas the absence of Panx1 in neurons reduces seizure manifestation²⁷.

GJ blockers to treat epilepsy

In a recent research Scemes et al.²⁸, they studied 13 GJ blockers as treatment options for epilepsy: CBX, quinine, mefloquine, quinidine, anandamide, oleamide, heptanol, octanol, meclofenamic acid, niflumic acid, flufenamic acid, glycyrrhetinic acid, and retinoic acid. In the in vitro experiments, all these compounds demonstrated to have anticonvulsant effects in brain slices. The blockers modified the behavioral parameters related to seizures induced by 4-aminopyridine, pentylenetetrazol, pilocarpine, penicillin, and maximal electroshock. Similar research works suggest that GJ blockers are a future alternative for the treatment of epilepsy. Nevertheless, most of these compounds have been discontinued as treatments due to their side effects, thus implying that further research must be conducted to identify the action mechanisms for neurological disorders such as epilepsy (Table 1).

Multiple studies on connexins have been conducted with the aim of understanding and reporting the devastating effects of connexin dysfunctions (Table 2). Connexins are important, since they form synapses. Moreover, GJs are present in all biological cells, except spermatozoids, and some types of blood cells.

AD

AD is the most common degenerative disease, in México, approximately 800,000 people have AD. Clinically, AD is caused by cerebral atrophy of the frontal cortex, along with neurofibrillary tangle and large numbers of senile plaques. The ApoE4 allele is usually seen as a risk factor for AD, yet health experts also look for other indicators. That is, not everybody carrying the ApoE4 gene variant will go onto develop AD as they grow old, although the probabilities are significantly higher. Moreover, some experts state that knowing that a person carries the ApoE4 allele does not benefit at all since none of the current treatment options cures the disease. ApoE gene polymorphic form ApoE4 (ε4) allele is the most commonly associated genetic risk factor linked with the late onset of the AD. In response to injury or neuroinflammation, Apoε4 undergoes neuron-specific proteolysis. Even if it is not clear the relationship between this allele and Cx43, it will be interesting to focus on Cx43 function under this condition²⁸. Thus far, the relationship between AD and GJs remains unclear, yet glial cells are thought to contribute to the aggressive propagation of this disease by inhibiting cell communication and promoting neuroinflammatory responses²⁹. Moreover, scientific evidence suggests a relationship between neurotoxicity of Aβ42 deposits in AD and upregulation of Cx43 as a result of released neurotoxic molecules that cause oxidative stress³⁰.

That said, researchers have found that Cx43 prevents, to some extent, the brain from deteriorating; however, Cx43 overexpression identified in experiments on AD-like pathologies³⁰ seems to severely affect intercellular communication, thus relating Cx43 overexpression with neuroinflammation.

Cx43 also seems to be involved in glutamate excitotoxicity induced by manganese exposure, thus promoting neurodegenerative diseases, such as AD and Parkinson's disease (PD). Moreover, in the relationship between GJ activity in glial cells and glutamate excitotoxicity, the latter is linked to excessive NMDAR activity and neurodegeneration³¹. That said, two promising treatments of AD include blocking specific GJs for Cx43³² and the Metabolic Enhancement for Neurodegeneration (MEND) approach. MEND is a 36-point therapeutic personalized program including aspects such as medication, comprehensive dietary changes, vitamins, brain stimulation, and exercise, to name but a few. There is a revision that mentions the importance of GJ in AD, their positive and negative role in neurons and other cell types, expressing that there is a need for further investigation in a GJ oriented treatment for AD³³.

PD

PD is characterized by the loss of dopaminergic neurons in the part of the brain known as the substantia nigra (SN). There are no exact numbers of Parkinson's patients in Mexico. However, the National Institute of Neurology and Neurosurgery estimates a prevalence of 50 new cases per 100,000 inhabitants/year. Clinical symptoms include shaking, rigidity or tremor, and motor disorders such as bradykinesia which means slowness of movement, and it is one of the cardinal symptoms of Parkinson's (i.e., difficulty with walking). The 1-methyl-4-fenyl-1,2,3,6-tetrahydropyridine (MPTP) chemical model is used to represent the conditions of PD, and it causes permanent symptoms. MPTP is extensively modeled in mice and is characterized by overexpression of Cx43 in both the striatum and the astrocytic hemichannels in the SN, thus contributing to the death of dopaminergic neurons³⁴. In addition, MPTP animal models have shown accelerated neuron loss in the absence of Cx30 as a result of a disruption in astrocyte energy metabolism³⁵.

A breakthrough in research on PD is the study of the inferior olivary nucleus (ION), which is a complex structure forming a bulge in the ventral surface of the medulla oblongata. The ION receives a wide range of sensory and motor afferents, and it is the source of the climbing fibers ascending to the cerebellum. Similarly, the ION is thought to play a key role in the generation of tremor in PD. Nowadays, olivary neurons are coupled by means of GJs, yet further research is necessary to determine their functional role. In other publications they mention the importance of focusing attention on the GJs for PD, for example, they say that GJ blocker treatment combined with inhibition of K+ channels might be a good approach to correct motor dysfunction in PD patients³⁶ and that the use of GJ blockers in PD appears to be a promising treatment, it has been reported to improve motor function in hemiparkinsonian rats since GJ activity elicits beta oscillations in the basal ganglia nuclei, leading to akinesia in PD³⁷.

Brain tumors (glioma)

A glioma is only one type of CNS tumor that originates in the glial cells of the brain or the spine. Gliomas are the most common type of brain tumors - they are space-occupying lesions causing intracranial pressure, vascular occlusion, and cerebral edema. In Mexico, an average of 30,000 new cases of brain cancer is detected annually, according to data provided by the Mexican Council of Neurological Surgery. The literature reports that in cases of severe gliomas, Cx43 is present in low concentrations; however, it is also known that the structure of Cx43 itself encourages glioma proliferation³⁸. In addition, Cx43 suppresses tumor growth, regardless of GJ channels. Namely, it is believed that Cx43 C-terminus directly inhibits tumor metastasis, possibly due to the correlation between Cx43 expression and protein kinase B (Akt)/extracellular signal-regulated kinase³⁹. In other words, Cx43 acts as an inhibitory regulator of the activation of growth factor receptors, usually related to treatments for glioblastomas (GBM).

The study of brain tumor cells entails a deep study of the cell cycle. For instance, preliminary data have shown that Cx43 helps regulate the cell cycle during the S phase by interacting with a protein kinase⁴⁰. However, Cx43 is also known to promote chemoresistance during GBM treatment by reducing the apoptotic effect of temozolomide⁴¹. From this perspective, researchers have been able to identify cancer cell lineages with these characteristics⁴². In fact, identifying cells with aberrant Cx43 expression allow experts to develop new treatment options for brain tumors and block GJs to prevent chemoresistance⁴³.

New approaches have been developed to identify tumor genesis response mechanisms. For instance, laser scanning microscopy (confocal laser scanning microscopy) and simple molecule localization microscopy are employed for the spatial localization of Cx43 in tumor genesis and the formation of metastasis⁴⁴. Moreover, the literature reports epigenetic approaches for Cx26, Cx30, and Cx43⁴⁵. In fact, cell homeostasis can help suppress gliomas, yet it is also theorized that tumor cells affect neighbor cells through GJs⁴⁶,⁴⁷, whereas increased GJ regulation decreases the proliferation of glioma cells⁴⁸.

Autism

Autism spectrum disorder (ASD) is a developmental disorder whose causes are usually unknown. It is influenced by multiple factors across race, ethnicity, and socioeconomic status aspects. According to the literature, in the USA, over 100 million families are affected by ASD. In 2017, around 1% of all children in Mexico, about 400,000 have autism. Recent evidence suggests that alterations of gut microbial-associated epitopes, including GJ alpha 1, can adversely affect the function of Cx43⁴⁹. Such results associate gastrointestinal problems in ASD with GJs and neuronal loss. In addition, research has found an increase in astrocytic Cx43 expression in a part of the brain's frontal cortex (i.e. Brodmann area 9) of subjects with autism, thus suggesting abnormal glial-neuronal communication in brains of ASD sufferers. Finally, studies on brain connectivity relying on electrophysiological techniques have used H. medicinalis as a key model to reduce the expression of specific connexins. In humans, this could help find a way to reduce overconnectivity in the brains of subjects with autism⁵⁰.

New studies have emerged to diagnose neuropathologies with different novel approaches. Histochemistry was used to proper identify neurons, neuritic processes and axons, myelin sheaths, neuroglial cells, and connective tissue in the nervous system⁵⁰. New strategies have been developed to predict ASD. One approach can be using genetic information contained in the Autism Genetic Resource Exchange; this database was used to predict the diagnostic of ASD in combination with an instrument of behavioral evaluation⁵¹. The other approach is the use of neuroimaging information which can be useful in the evaluation of psychiatric disorders. For ASD computational techniques were used to process magnetic resonance images of subjects with ASD and control groups⁵²,⁵³. There is no relationship between neuroimaging and GJs. Neuroimaging can be used as alternative information to try to evaluate the condition of a patient. Magnetic resonance procedures can be helpful to see the brain activity of patients with ASD. Moreover, computational techniques can be useful to process that information.

Symptom onset, genetic, and neuropathological data were examined from patients with Lewy body proteins to determine the relationship between those proteins and AD⁵⁴. In addition, postmortem magnetic resonance was used to examine serial coronal sections, horizontal sections of brainstem, and cerebellum to find neuropathological lesions⁵⁴,⁵⁵.