The NCX3 gene amplifies pain signals within the spinal cord


Oxford researchers have discovered a gene that regulates pain sensitization by amplifying pain signals within the spinal cord, helping them to understand an important mechanism underlying chronic pain in humans and providing a new treatment target.

Chronic pain is a common issue affecting millions of people worldwide, but why some people are more prone to it and what factors lead to chronic pain are not fully understood.

It is well known that repeated stimulation, such as with a sharp pin prick, can lead to a heightened sensitivity to pain. This process is called “pain wind-up” and contributes to clinical pain disorders.

In a two-part study, researchers from Oxford’s Nuffield Department of Clinical Neurosciences first compared genetic variation in samples from more than 1,000 participants from Colombia, to look for clues as to whether there were any genetic variants more common in people who experienced greater pain wind-up. They noted a significant difference in variants of one specific gene (the protein Sodium Calcium exchanger type-3, NCX3).

The researchers then undertook a series of experiments in mice, to understand how NCX3 regulates pain wind-up and whether it may be a treatment target. NCX3 was expressed in the mouse spinal cord neurons that process and transmit pain signals to the brain.

NCX3 was needed by these neurons to export the excess calcium that builds up following activity. In the absence of NCX3 the spinal cord neurons showed more activity in response to injury signals from the periphery and pain wind-up was increased.

Conversely, increasing the levels of NCX3 within the spinal cord could reduce pain in the mouse.

David Bennett, professor of neurology and neurobiology of the Nuffield Department of Clinical Neuroscience, said: “This is the first time that we have been able to study pain in humans and then to directly demonstrate the mechanism behind it in mice, which provides us with a really broad understanding of the factors involved and how we can begin developing new treatments for it.”

Professor Bennett added: “Chronic pain is a global problem, and can be immensely debilitating. We carried out the study in Colombia because of the mixed ancestry of the population there, including Native Indian, African and European populations, which gave us a broad range of genetic diversity to look at. This makes these findings so exciting because of their potential international applications.

“The findings imply that any drugs which can increase activity of NCX3 would be predicted to reduce pain sensitization in humans.”

The results of the present study demonstrate that mitochondrial dysfunction occurring in mesencephalic neurons obtained from A53T-α-syn mice is associated with dopaminergic neuronal demise and consequently to a late activation of neuroinflammation in the nigrostriatal pathway.

Moreover, these results also confirm the potential role of NCX3 in triggering mitochondrial dysfunction and neuroinflammatory response in a PD animal model. Indeed, in the striatum obtained from WT and A53T-α-syn mice no differences in NCX3 protein expression are detected during aging, and mitochondrial function is preserved.

This finding is in line with data previously reported [20], showing that NCX3, apart its localization at plasma membrane level, is also localized on the outer mitochondrial membrane and promotes mitochondrial calcium efflux in physiological and pathological conditions, thus playing a pivotal role in the maintenance of intracellular Na+ and Ca2+ homeostasis not only in brain ischemia but also in neurodegenerative diseases [17,36,37,45,46,47,48,49].

More interesting, the finding that NCX3 is differently expressed in dopaminergic neurons in the midbrain compared to striatum led us to speculate a new potential mechanism contributing to the selective neuronal degeneration observed in PD. This result allows us to exclude the hypothesis that the increasing load of aggregated pathological form of α-synuclein, described in this model [39,40,42] might have a detrimental role through the regulation of NCX3 activity and expression.

Indeed, due to their pacemaking function, mesencephalic neurons are more frequently exposed to continued calcium transients that make them more prone to mCa2+ overload and, consequently, to mitochondrial dysfunction [13,50,51,52,53]. In this scenario, the impairment of the expression and activity of NCX3 in mesencephalic neurons might contribute to their selective vulnerability in the midbrain of A53T-α-syn mice, due to NCX3′s contribution to the perturbation of [Ca2+]i concentration, as already hypothesized by Sirabella et al. (2018) [22].

Therefore, the experiments performed in the present study further confirm that NCX3-dependent mitochondrial dysfunction occurring in mesencephalic neurons from A53T-α-syn might be responsible for the neuronal damage with consequent late activation of the cellular events along the nigrostriatal pathway that, in turn, might be responsible for PD progression. Indeed, Western blot experiments performed in 4 and 16-month-old A53T-α-syn and WT mice showed an increase in Cyt c and nNOS protein expression, both markers of mitochondrial damage and oxidative stress, respectively, that was already detectable in the midbrain of 4-month-old A53T-α-syn mice and was still elevated in the late stage of disease compared to WT mice.

Conversely, in the striatum of A53T-α-syn mice, in which NCX3 expression did not change, in comparison to WT mice, an increase in Cyt c and nNOS protein levels occurred only in the late stage of the disease. This is in line with previously described immunohistochemistry experiments performed in 12-month-old A53T-α-syn mice showing a reduction in TH expression in the midbrain of these mice compared to WT [22].

Therefore, the results of the present study suggest that a progressive impairment of the oxidative metabolism occurring in the early stage in mesencephalic neurons might be claimed as a pathogenetic mechanism leading to dopaminergic neuronal damage responsible for the late progression of PD. Indeed, no increase in pro-inflammatory proteins was detected in 4-month-old A53T-α-syn both in the midbrain and in the striatum (data not shown), although mitochondrial dysfunction occurred.

This finding supports the data recently reported in the literature that correlate mitochondrial dysfunction to the triggering of the neuroinflammatory process in PD. Indeed, damaged mitochondria can release numerous pro-inflammatory factors which, triggering microglial activation, lead to neuroinflammation in the nigrostriatal pathway [54,55,56].

This hypothesis is confirmed by the results presented in this study regarding the increase in IBA-1, IL-1β and iNOS detected in the striatum of 16-month-old A53T-α-syn mice, in which an increase in Cyt c and nNOS occurs. Moreover, the activation of microglial cells in the striatum of aged A53T-α-syn mice is associated with an increase in GFAP, thus confirming a relationship between mitochondrial-induced neuroinflammation and glial activation [57,58,59,60].

Conversely, in the midbrain of 16-month-old A53T-α-syn mice the increase in Cyt c and nNOS protein expression is accompanied by glial proliferation, as confirmed by the increase in GFAP protein levels without microglial activation. These findings, in accordance with data previously reported in 12-month-old A53T-α-syn mice [22], further support the hypothesis that in the midbrain, the activation of glial cells might be a consequence of the cellular responses triggered into the striatum of transgenic mice, and in turn, it might further contribute to dopaminergic neuronal demise observed in the midbrain during PD progression.

Moreover, the data obtained in the striatum also suggest that the molecular mechanisms involved in the activation of microglial cells are different from those occurring in astrocytes. In fact, it is possible to hypothesize that the neuronal damage related to the impairment in mitochondrial membrane permeability in the midbrain since the early stages of the disease might promote the release of neuronal toxic factors able to stimulate the activation of microglial cells in the striatum of A53T-α-syn adult mice.

Once activated, microglial cells can release pro-inflammatory cytokines in adult mice and in turn, promote astroglia proliferation in the striatum and in the midbrain, as confirmed by the increase in NCX1 protein expression described in these two brain areas in aged A53T-α-syn mice (Figure 5).

Interestingly, in astrocytes obtained from midbrain of A53T-α-syn mice mitochondria, although they contain higher calcium compared to WT cells, they are not depolarized, thus suggesting their ability to support cellular proliferation. These findings let us hypothesize that the normal expression of NCX1 in transgenic astrocytes by maintaining [Ca2+]i within physiological range is able to preserve mitochondrial function and, consequently, to promote gliosis in adult mice, despite the impairment of NCX3 protein expression. In this scenario, NCX1 overexpression would not be directly related to a detrimental effect in glial cells.

This is in line with data previously reported in the ischemic brain [33] and might explain the consequent increase in dopaminergic neuronal injury observed in the midbrain of A53T-α-syn aged mice (Figure 5). On the other hand, the increased NCX1 expression occurring in striatum of A53T-α-syn mice being a reflection of glial proliferation might represent a useful druggable target to modulate neuroinflammation in PD.

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Figure 5
Schematic model illustrating the cellular and molecular events leading to neuroinflammation in A53T-α-syn mice. (A) The NCX3 impaired expression in the midbrain leads to mitochondrial dysfunction in dopaminergic neurons with consequent cellular damage and release of toxic factors (probably aggregated α-syn) able to induce microglial cells’ activation in the striatum. (B) The microglial activation in the striatum may induce pro-inflammatory cytokines’ release that in turn stimulates NCX1-driven astrocyte proliferation. Finally, the activated astrocytes in the striatum may invade the midbrain, further contributing to dopaminergic neuronal damage.

In conclusion, the results reported in the present study demonstrate that mitochondrial dysfunction in dopaminergic neuronal cells might exert a detrimental role in PD progression. Indeed, mitochondrial dysfunction occurring in mesencephalic neurons in A53T-α-syn mice at the early stage of the disease promotes neuronal degeneration and activates microglial cells in the striatum. The activated microglia can in turn promote pro-inflammatory factors’ release in the striatum of these mice with consequent glial activation and progressive impairment of dopaminergic neuronal plasticity in the late stage of the disease.

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Source: University of Oxford


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