1. Presented by: Justin P. Smith
Epigenetic Gene Silencing Underlies C-Fiber Dysfunctions in Neuropathic Pain
Presented by: Justin P. Smith

2. Background Peripheral nerve damage causes neuropathic pain
Paradoxical sensations of + and – symptoms
+ symptoms: hyperalgesia, allodynia, paresthesia
- symptoms: hypoesthesia, hypoalgesia
Negative symptoms NOT extensively investigated

3. Terms:
Hypoesthesia: A reduced sense of touch or sensation, or a partial loss of sensitivity to sensory stimuli
Hypoalgesia: A decreased sensitivity to painful stimuli
Allodynia: A stimulus which does not normally provoke pain, either thermal or mechanical, often occurs after injury to a site.
Hyperalgesia: an extreme reaction to a stimulus which is normally painful
Parethesia: tingling or pricking (pins and needles)

4. Negative Symptoms
Possible mechanisms:
Dysfunction of small-diameter C-fibers
Loss of C-fiber terminals
Impairment of C-fiber mediated axon-reflex flare responses
Increase in the threshold against C-fiber-specific-stimuli

5. /NRSF
Different possible outcomes of re-expression of REST in mature neurons. (a) Chromatin status of mature neurons, in the absence of REST. Active neuronal chromatin is in a relaxed conformation and marked by an increased amount of trimethylated lysine 4 on histone 3 (tri-mK4) compared with the amount of dimethylated lysine 4 on histone 3 (di-mK4). REST reassembles a core corepressor complex. (b) Re-expression of REST might result in reprogramming of neuronal genes to a repressed state by reduction of trimethylation of lysine 4 on histone H3 (tri-mK4), and compaction, probably by histone deacetylation, of chromatin. (c) Alternatively, REST recruits corepressors, but is unable to reprogram the chromatin to a repressed state (number of trimethyl lysine 4 residues is unchanged from original neuronal chromatin and chromatin stays relaxed) and, therefore, neuronal genes are still transcribed.
Reprogramming of neuronal genes to a repressed state by reduction of trimethylation of lysine 4 on histone H3 (tri-mK4), and compaction, probably by histone deacetylation, of chromatin
Current Opinion in Neurobiology Volume 15, Issue 5, October 2005, Pages Neuronal and glial cell biology / New technologies

6. Histone Gene Activated Gene Repressed Acetyl groups Methyl groups
Histone Acetyltransferase (HAT)
Histone Deacetylase (HDAC)
Acetyl groups
Gene Activated
Histone
Gene Repressed
Methyl groups
Histone Methyltransferase (HMT)
Histone Demethylase (HDM)

7. Claims!
Nerve injury – long lasting neuron-restrictive silencer factor (NRSF) in DRG causing EPIGENETIC SILENCEING of MOP gene
Loss of pharmacological target for peripheral morphine analgesia
Possible EPIGENETIC SILENCEING of Na 1.8 gene, unique NRSE sequences

8. Methods Male C57BL/6J mice 20–25 g, food & water ad libitum
Sciatic nerve was partially ligated
Oligonucleotide treatments
Nociception test
Drug treatments
Quantitative real-time PCR
Western blot analysis
Immunohistochemistry
Chromatin immunoprecipitation
(ChIP) assays

10. Design
Wanted validation that MOP and Na 1.8 would be downregulated at transcriptional level
Isolated L4-6 dorsal root ganglion at days 1, 3, 7 and 14 after sciatic nerve was partially ligated
Checked mRNA expression of MOP and Na 1.8 using rt-PCR

11. Fig 1 21 bp sites Forward Reverse
Nerve injury causes a long-lasting reduction in MOP and Nav1.8 mRNA levels in the DRG, starting from days 1 and 3 post injury and the downregulations persisted at least 14 d post injury
21 bp sites
Forward
Reverse

12. Fig 1
Threshold of 80 TFSEARCH

13. Epigenetic upregulation of NRSF expression post nerve injury
NRSF expression regulates silencing activity
Examined NRSF expression in DRG after nerve injury, again on days 1, 3, 7 and 14
Exon II had most prominent induction (compared to EI and EIII)
Looked at H3 and H4- both correlated with transcriptional activation at NRSF promoter II upstream of exon II
Wanted to check NRSF protein expression – western blot using NRSF-antibody
Stained for NRSF and NeuN (neuronal marker)

14. Fig 2

15. Fig 2
ChIP analysis: nerve injury causes a robust increase in the acetylation of histone H4, but not of H3, in the NRSF promoter II region

16. Fig 2
Western
NRSF-protein increase on day 7

17. NRSF-RED, NeuN-Green Location: DRG

18. NRSF binds to NRSE sites of MOP & Na 1.8 post nerve injury day 7
NRSE serve as NRSF-binding sites
ChIP analysis- NRSF binding to NRSE of MOP and Na 1.8
ChIP analysis- acetylation of H3 and H4 in NRSE sequences of MOP and Na 1.8 genes (in L4-6 of DRG on day 7)
Included sharm and injury surgeries

19. Fig 3 rtPCR Nerve injury caused a drastic increase in NRSF
binding to MOP-NRSE, Nav1.8-NRSE-1,
and Nav1.8-NRSE-2 sequences: suggests NRSE sequences capable of serving as NRSF-binding sites.

20. Fig 3 PCR-targeted regions
Scanning ChIP analysis of acetylation levels of histone H3 (AcH3) and H4 (AcH4)
in the genomic regions spanning NRSE sequences within MOP

21. Data suggests that nerve injury
induces repressive chromatin states
around the NRSE sequences of MOP and
Nav1.8 genes through NRSF-HDAC-mediated
mechanisms
Fig 3
Scanning ChIP analysis of acetylation levels of histone H3 (AcH3) and H4 (AcH4)
in the genomic regions spanning NRSE sequences Nav1.8 genes

22. AS-ODN and MS-ODN NRSF knock-down by AS-ODN
To check if AS-ODN (via Dicer enzyme) will decrease NRSF and block nerve injury downregulation of MOP and Na 1.8 (levels stay elevated)
How?
Injected Antisense or Mismatch intrathecally on 1st, 3rd and 5th days then nerve injury, again injected days 1, 3, 5, 6 post injury
Assessed day 7 post injury

23. Fig 4 Can NRSF contribute
to downregulation of MOP and Nav1.8 genes after nerve injury? Pretreated intrathecally with an AS-ODN against NRSF or a corresponding MS-ODN
Western blot analysis: NRSF protein levels in
the DRG were markedly reduced by ASODN,
but not by MS-ODN

24. Fig 4 AS-ODN significantly blocked the nerve
injury-induced downregulation of MOP
and Nav1.8
Suggest that NRSF-mediated mechanisms
are responsible for the transcriptional
suppression of MOP and Nav1.8 genes in the DRG after nerve injury
NO EFFECT

25. Fig 4 Genes for other receptors, TFSEARCH scores <80.
AS-ODN blocked downregulations
TRPM8 and TRPA1, which have been proposed to function as cold receptors

26. C-fiber function by NRSF knockdown
Examined pain behavior: C-fiber hypoesthesia (numbness), A-fiber hypersensitization, thermal hyperalgesia (extreme reaction to pain), and mechanical allodynia (thermal or mechanical stimuli that doesn’t normally produce pain)
Electrical stimulation-induced paw withdrawl (EPW)-electrodes on paw
Thermal paw withdrawl test
Mechanical paw pressure test
A (a selective Na 1.8 blocker) administered

27. Abnormal pain behaviors in AS-ODN-treated mice,
Fig 5
Hypoesthesia
(reduced sense)
hypersensitization
Sine-wave
Abnormal pain behaviors in AS-ODN-treated mice,

28. Fig 5
Recovery of nerve injury-induced loss of peripheral A hypoesthesia by AS-ODN. The C-fiber responses were assessed 30 min after intraplantar injection of control (Cont) or A (30 nmol).
Fig 3S

29. AS-ODN ameliorates the loss of peripheral morphine analgesia
Thermal paw withdrawl test- morphine interaction
AUC- area under curve

30. Fig 6
AS reduced MOP expression, increase in pain during thermal paw withdrawal test.
Consistent with Fig 1A and Fig 4B

31. Discussion
Nerve injury caused an epigenetic induction of NRSF gene expression in the DRG neurons
Nerve injury induces histone hypoacetylation at NRSE sequences within MOP and Nav1.8 genes with an increase in direct NRSF binding
The antisense knockdown of NRSF significantly blocked nerve injury-induced transcriptional repression of MOP, Nav1.8, TRPM8, and TRPA1, but not CGRP, genes in the DRG
All of the nerve injury-induced C-fiber hypoesthesia and the losses of peripheral A hypoesthesia and peripheral morphine analgesia were markedly recovered by NRSF knockdown

32. Thank You