17 May 2013. One of the focal points of schizophrenia research over the past decade has been abnormalities detected in a subgroup of prefrontal cortex interneurons distinguished by the fact that they contain the molecule parvalbumin. A new study published May 13 in the Proceedings of the National Academy of Sciences finds that these neurons appear to have protective shields against oxidative stress, suggesting a mechanism that could go awry in schizophrenia.
The study, led by Kim Do of the University of Lausanne, Switzerland, explored the function of neuron-swathing sheaths called “perineuronal nets” (PNNs) in mice. The researchers found that PNNs surrounding parvalbumin-containing (PV+) interneurons limited their levels of oxidative stress, and that degrading the PNNs led to increased oxidative stress and desynchronized brain activity.
The findings highlight the workings of PV+ interneurons, which have been implicated in schizophrenia by a number of studies. Postmortem studies have found that PV+ interneurons are low on GAD67, the enzyme that makes the inhibitory neurotransmitter γ-aminobutyric acid (GABA), as well as PV itself (Lewis et al., 2012). These inhibitory neurons emit rapid-fire action potentials and are key controllers of synchronous activity in the brain, including the γ waves linked to working memory which are affected by schizophrenia. Electroencephalogram (EEG) studies find degraded synchrony in schizophrenia, possibly reflecting weakened inhibitory signaling in the brain.
The new study suggests that the extracellular matrix molecules that comprise PNNs surrounding PV+ interneurons have a hand in compromising their function. Fewer PNNs have been found in a postmortem study of schizophrenia (see SRF related news story), and clues from other research have suggested that a PNN’s meshwork of protein and carbohydrate molecules provides more than just a place for a neuron to sit. PNNs have been suggested to stabilize synapses, to end critical periods during development (Kwok et al., 2011), and to offer a measure of protection against oxidative stress (Morawski et al., 2004). The new study supports the last idea and suggests that protection by PNNs is particularly important to PV+ interneurons, which may be more vulnerable to oxidative stress because their fast spiking comes with a high metabolic demand.
Intense firing in PV+ interneurons can generate reactive oxidative molecules within the cell. At sufficiently high levels, this can damage the cell’s innards, including its DNA. To guard against this, cells have other “antioxidant” molecules to sop up these reactive oxygen species. First authors Jan-Harry Cabungcal and Pascal Steullet explored their hypothesis that PNNs shield PV+ interneurons from oxidative stress using mice genetically engineered to be especially vulnerable to oxidative stress. These mice lacked a subunit of an enzyme called glutamate cysteine ligase (Gclm), which produces glutathione, an antioxidant. Gclm knockouts have lower than usual glutathione, akin to what Do and colleagues have previously found in schizophrenia (Do et al., 2000). Similarly, they have reported a genetic association between the GCLM gene and schizophrenia (Tosic et al., 2006), suggesting a role for oxidative stress in the disorder (see SRF related news story).
Using a product of DNA oxidation as a marker of oxidative stress, the researchers found higher levels of oxidative stress in neurons of the anterior cingulate cortex of Gclm knockouts compared to wild-type mice; however, the number of PV+ interneurons and PNNs detected were the same between the two groups at postnatal day 90. But growing up under chronic oxidative stress eventually took its toll: by postnatal day 180, the researchers counted fewer PV+ interneurons, but the same number of PNNs, compared to wild-type mice. This suggested to them that the lost PV+ interneurons may have been those without good PNNs.
The researchers found further support for this when they did a cell-by-cell accounting of how well oxidative stress correlated with the integrity of the PNN. For this experiment, they acutely increased oxidative stress further, using a dopamine reuptake inhibitor called GBR that increases reactive oxygen species, in addition to increasing extracellular dopamine. Under these conditions, the degree of DNA oxidation label was inversely correlated with PNN density (r = 0.42, P = 0.0032), which suggests that the better the PNN surrounding a cell, the less oxidative stress felt by the cell.
Stripping away the nets
The researchers also noticed that GBR treatment brought on a decrease in PNN label around PV+ interneurons in Gclm knockouts, but not in controls, suggesting that the PNN meshwork itself is sensitive to oxidative stress. This was further supported in a conditional knockout experiment using mice that lacked the glutathione-making enzyme only in PV+ interneurons: substantially lower numbers of both PV+ interneurons and PNNs were found, compared to wild-type mice. This indicates that oxidative stress inside a cell can degrade PV expression within the cell and PNN integrity outside of the cell, but it remains unclear whether PNN degradation comes before or after PV reduction.
Finally, the researchers stripped neurons of their PNNs with an enzyme called chondroitinase ABC (ChABC) injected in one side of the anterior cingulate cortex of Gclm knockouts. Following this with 11 days of GBR treatment, the researchers found a significant decrease in PV+ interneurons and a concomitant increase in oxidative stress marker in the ChABC injected side compared to the sham-injected hemisphere. The researchers did not find a decrease in calbindin and calretinin types of interneurons, however, which argues that PNNs are specific to the PV+ type of interneuron.
Taking brain slices from these animals, the researchers found that the rhythmic activity induced from ChABC-treated hemispheres was weaker: specifically, the power of oscillations in the frequency of the β and γ bands (13-28 Hz and 30-60 Hz, respectively) was decreased in the ChABC-treated hemispheres of GBR treated Gclm knockouts compared to the sham-injected hemispheres. The combination of PNN loss and oxidative stress seemed necessary to reduce oscillations in this range because when this experiment was done in wild-type mice, the ChABC-treated hemispheres had higher levels of β and γ oscillations.
Future work will have to assess whether these nets actually protect PV+ interneurons from oxidative stress in schizophrenia. Although a clear role for oxidative stress in schizophrenia is not yet settled, the findings illustrate how seemingly innocuous changes to extracellular matrix molecules can translate into widespread changes in brain activity.—Michele Solis.
Cabungcal JH, Steullet P, Morishita H, Kraftsik R, Cuenod M, Hensch TK, Do KQ. Perineuronal nets protect fast-spiking interneurons against oxidative stress. Proc Natl Acad Sci U S A. 2013 May 13. Abstract