Molluscan memory of injury: evolutionary insights into chronic pain and neurological disorders.

Molluscan preparations have yielded seminal discoveries in neuroscience, but the experimental advantages of this group have not, until now, been complemented by adequate molecular or genomic information for comparisons to genetically defined model organisms in other phyla. The recent sequencing of the transcriptome and genome of Aplysia californica, however, will enable extensive comparative studies at the molecular level. Among other benefits, this will bring the power of individually identifiable and manipulable neurons to bear upon questions of cellular function for evolutionarily conserved genes associated with clinically important neural dysfunction. Because of the slower rate of gene evolution in this molluscan lineage, more homologs of genes associated with human disease are present in Aplysia than in leading model organisms from Arthropoda (Drosophila) or Nematoda (Caenorhabditis elegans). Research has hardly begun in molluscs on the cellular functions of gene products that in humans are associated with neurological diseases. On the other hand, much is known about molecular and cellular mechanisms of long-term neuronal plasticity. Persistent nociceptive sensitization of nociceptors in Aplysia displays many functional similarities to alterations in mammalian nociceptors associated with the clinical problem of chronic pain. Moreover, in Aplysia and mammals the same cell signaling pathways trigger persistent enhancement of excitability and synaptic transmission following noxious stimulation, and these highly conserved pathways are also used to induce memory traces in neural circuits of diverse species. This functional and molecular overlap in distantly related lineages and neuronal types supports the proposal that fundamental plasticity mechanisms important for memory, chronic pain, and other lasting alterations evolved from adaptive responses to peripheral injury in the earliest neurons. Molluscan preparations should become increasingly useful for comparative studies across phyla that can provide insight into cellular functions of clinically important genes.

DNA incisions stimulate genetic instability of triplet repeat sequences related to human hereditary neurological diseases

The molecular mechanisms responsible for the expansion and deletion of trinucleotide repeat sequences (TRS) are the focus of our studies. Several hereditary neurological diseases including Huntington's disease, myotonic dystrophy, and fragile X syndrome are associated with the instability of TRS. Using the well defined and controllable model system of Escherichia coli, the influences of three types of DNA incisions on genetic instability of CTG•CAG repeats were studied: DNA double-strand breaks (DSB), single-strand nicks, and single-strand gaps. The DNA incisions were generated in pUC19 derivatives by in vitro cleavage with restriction endonucleases. The cleaved DNA was then transformed into E. coli parental and mutant strains. Double-strand breaks induced deletions throughout the TRS region in an orientation dependent manner relative to the origin of replication. The extent of instability was enhanced by the repeat length and sequence (CTG•CAG vs. CGG•CCG). Mutations in recA and recBC increased deletions, mutations in recF stabilized the TRS, whereas mutations in ruvA had no effect. DSB were repaired by intramolecular recombination, versus an intermolecular gene conversion or crossover mechanism. 30 nt gaps formed a distinct 30 nt deletion product, whereas single strand nicks and gaps of 15 nts did not induce expansions or deletions. Formation of this deletion product required the CTG•CAG repeats to be present in the single-stranded region and was stimulated by E. coli DNA ligase, but was not dependent upon the RecFOR pathway. Models are presented to explain the DSB induced instabilities and formation of the 30 nucleotide deletion product. In addition to the in vitro creation of DSBs, several attempts to generate this incision in vivo with the use of EcoR I restriction modification systems were conducted. ^