Aim of Investigation:

Pathological states of neuropathic pain are related to sensitization of deep dorsal horn neurons that are likely to amplify nociceptive transmission, causing allodynia and hyperalgesia. The efficiency of GABAb agonists in the treatment of chronic pain is controversial. The present project aims to investigate possible dysfunction of GABAb inhibitory control of pain sensitization in the spinal cord of animal models.

Our working hypothesis is that functional alteration of heterodimeric GABAb receptor depends on its dissociation, possibly triggered by intracellular partner proteins such as 14.3.3.

Methods:

We investigated the co-distribution of GABAb1 and 14.3.3 with microscopy techniques. The physical interactions between the various partner proteins have been studied with FRET/FLIM imaging and with co-immunoprecipitation approaches. The functional outcome of these interactions have been assessed in vitro with patch-clamp techniques, and in vivo with behavioural tests.

Results:

Our results showed that in a rat neuropathic pain model, 14.3.3 expression is up-regulated in the dorsal horn. GABAb receptor and 14.3.3 are codistributed in the same spinal neurons. The relative distribution of GABAb and 14.3.3 in spinal cord, investigated by electron microscopy, showed that the subcellular colocalization between GABAb1 and 14-3-3 is increased after spinal nerve ligation.

Moreover, in cultures of spinal neuron, 14.3.3 overexpression resulted in a partial loss of colocalization between GABAb1 and b2 subunits, suggesting that 14.3.3 induced the dissociation of the GABAb receptor. A FRET-based imaging approach further confirmed the dynamic of 14.3.3-GABAb1 interactions in neurons.

In COS cell culture, immunoprecipitation experiments demonstrated the physical association between 14.3.3 and GABAb1 subunit. Furthermore, it ensured the effects of 14.3.3 overexpression in disrupting the GABAb heterodimer.

To reverse GABAb dysfunction in the dorsal horn, we designed different strategies based on siRNA-mediated 14.3.3 knock-down and on the use of synthetic peptides that block 14.3.3 binding site on GABAb1 subunit. The behavioural assessment of mechanical allodynia revealed that both strategies are efficient in potentiating the inhibitory effect of intrathecally-injected Baclofen in neuropathic rats.

Conclusions:

By targeting associated proteins, it may thus be possible to improve the pharmacological efficiency of GABAb agonists in the treatment of neuropathic pain.