Research articleEffects of the dimeric PSD-95 inhibitor UCCB01-144 on functional recovery after fimbria-fornix transection in rats
Introduction
Glutamate is the most abundant excitatory neurotransmitter in the human central nervous system, and excessive levels of glutamate and stimulation of glutamatergic receptors have been linked to neuronal dysfunction and degeneration in a broad spectrum of neurological disorders including stroke and traumatic brain injury (TBI) (Lau and Tymianski, 2010, Kostandy, 2012). Numerous cellular mechanisms contribute to glutamate mediated cell death, also known as glutamate excitotoxicity, but generation of neurotoxic levels of the signaling molecule nitric oxide (NO) is considered central to the process (Dawson and Dawson, 1996). In neurons NO is synthesized by the calmodulin-dependent enzyme neuronal nitric oxide synthase (nNOS) (Guix et al., 2005), which is linked to the N-methyl-d-aspartate-type of ionotropic glutamate receptors (NMDAR) via the post-synaptic density protein of molecular weight 95 kDa (PSD-95) (Christopherson et al., 1999). PSD-95 binds the C-terminus of the GluN2B-subunit of NMDARs and the N-terminus of nNOS via its first two PSD-95/Discs large/Zona-occludens-1-(PDZ) domains thus coupling NMDAR activity to NO formation (Christopherson et al., 1999, Sattler et al., 1999). In this way, calcium influx through NMDARs preferentially leads to activation of nNOS, and neurotoxic levels of NO may be produced when NMDARs are overstimulated due to excessive extracellular glutamate (Sattler et al., 1999, Aarts et al., 2002).
Pharmacological inhibition of the NMDAR/PSD-95/nNOS-interaction with the monomeric PSD-95 inhibitor NA-1 (also known as Tat-NR2B9c) has been reported to be neuroprotective in numerous studies of experimental stroke in rodents and non-human primates as well as in a phase II clinical trial in humans with iatrogenic stroke (Montaner et al., 2013). We have recently designed and synthesized dimeric inhibitors that simultaneously bind the tandem PDZ1-2 domains of PSD-95 and show increased blood plasma stability and affinity compared to NA-1 (Bach et al., 2012). The dimeric PSD-95 inhibitor UCCB01-144 (also known as Tat-N-Dimer) transcends the blood-brain barrier and is able to reduce infarct volumes with up to 40% and improve motor functioning, when injected 30 min after permanent medial cerebral artery occlusion in mice (Bach et al., 2012). PSD-95 inhibitors may thus be promising pharmacological agents in the treatment of brain injury including TBI (Shear et al., 2011, Shear et al., 2009), which has pathophysiological mechanisms in common with stroke (Bramlett and Dietrich, 2004).
Thus far PSD-95 inhibitors have only been investigated to a limited extent in TBI; however we recently found that UCCB01-144 (Sommer et al., 2017a) and the analogous compound UCCB01-147 (Sommer et al., 2017b) did not provide neuroprotection in the controlled cortical impact (CCI) model of TBI in rats. However, no single animal model is currently able to model the heterogeneous and complex pathophysiology of TBI, and CCI is primarily a model of focal contusional injury (Marklund and Hillered, 2011). Injury mechanisms, biomarkers and behavioral deficits have been reported to vary between the most commonly used animal models of TBI (Mondello et al., 2016), and in order to elucidate whether treatment effects can be generalized or are related to specific pathophysiological mechanisms and sub-types of TBI, it is necessary to assess treatments in different models of TBI (Kochanek et al., 2016).
The aim of this preliminary study was to further investigate the neuroprotective abilities of UCCB01-144 by administering the compound after bilateral transection of the fimbria-fornix (FF). FF-transection may be described as a model of focal axotomy and results in excitotoxic cell death and significant anterograde and retrograde neurodegeneration in the hippocampus and cholinergic septal nuclei (Cassel et al., 1997, Ginsberg et al., 1999, Ginsberg and Martin, 2002). Focal axotomy is a type of injury that may be seen in addition to other injuries after TBI, and in human patients hippocampal and fornix atrophy are prominent and correlate with injury severity (Tate and Bigler, 2000, Ariza et al., 2006, Tomaiuolo et al., 2004). Contrary to stroke, FF-transection is only associated with minor direct vascular consequences, and beneficial effects of PSD-95 inhibitors in this experimental paradigm may thus indicate a broader therapeutic potential and application of these pharmacological agents in brain injury.
In the current study, we administered UCCB01-144 immediately following FF-transection or sham surgery in rats and monitored the effects of treatment on spatial delayed alternation in a T-maze, which is a task that primarily reflects working memory, egocentric spatial orientation and response flexibility (Mogensen et al., 2007, Dudchenko, 2004). We have previously demonstrated that spatial delayed alternation is sensitive to FF-transection and pharmacological as well as non-pharmacological interventions in rats (Mogensen et al., 2007, Mogensen et al., 2008, Gram et al., 2015).
Section snippets
Animals and ethics statement
Thirty two male Wistar rats (HanTac:WH) obtained from Taconic, Denmark were used for the experiments. At the time of surgery animals had a mean age of 14 weeks (range 13–15 weeks) and a mean body weight of 337 g (range 297–366 g). Animals were pair-housed in Makrolon type 3 H cages with ad libitum access to water in a colony room with a 12-hour light cycle (lights on from 19.00 to 07.00 h), and temperature set to 22 ± 2 °C. Standard enrichment in all cages consisted of plastic shelters, bedding and
Spatial delayed alternation
As expected there were marked differences between FF-transected and sham groups during the first sessions of spatial delayed alternation with the former groups making nearly twice as many errors as the sham groups on average (Fig. 1). There was a significant main effect of session indicating that all groups exhibited learning in terms of a reduced number of errors over time (F6,168 = 74.259, p < 0.001), although learning curves appeared to stabilize or stagnate from session 12 and onwards in sham
Discussion
In this preliminary study no neuroprotective effects of the dimeric PSD-95 inhibitor UCCB01-144 were evident after bilateral FF-transection. On the contrary, there was a tendency for decelerated learning in animals given UCCB01-144 after FF-transection resulting in flatter learning curves and a greater number of errors during spatial delayed alternation. However, differences between FF/-144 and FF/Vehicle groups failed to reach statistical significance, which may at least in part be ascribed to
Abbreviations
- ANOVA
analysis of variance
- ATP
adenosine 5′-triphosphate
- CaMKIV
calmodulin kinase IV
- CCI
controlled cortical impact
- CREB
cAMP Response Element Binding protein
- FF
fimbria-fornix
- IP
intraperitoneal
- IV
intravenous
- LTD
long-term depression
- LTP
long-term potentiation
- NMDA
N-methyl-d-aspartate
- NMDAR
N-methyl-d-aspartate-type receptors
- nNOS
neuronal nitric oxide synthase
- NO
nitric oxide
- NOS
nitric oxide synthase
- NOX2
NADPH oxidase
- PDZ
PSD-95/Discs large/Zona-occludens-1
- PFC
prefrontal cortex
- PSD-95
post-synaptic density protein of
Acknowledgments
We gratefully acknowledge and are indebted to Johan Høy Jensen, Kiva Maria Krohn, Christina Rytter Vilsen, Heidi Marie Nielsen and Shahrokh Khazraie Padrah for their valuable assistance and technical expertise.
Conflict of interest statement
The authors declare the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: AB and KS are co-founders of Avilex Pharma that develops inhibitors of PDZ domain proteins including UCCB01-144. No other potential competing financial interests were reported by any of the authors.
Data accessibility statement
All experimental data will be made available upon request.
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- 1
Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 162, DK-2100 Copenhagen, Denmark.
- 2
The Unit for Cognitive Neuroscience (UCN), Department of Psychology, University of Copenhagen, Øster Farimagsgade 2A, DK-1353 Copenhagen, Denmark.
- 3
Department of Drug Design and Pharmacology, University of Copenhagen, Jagtvej 160, DK-2100 Copenhagen, Denmark.