“Genetic Characterization of the progressive hydrocephaly (prh) Mouse Mutant”
Shawn Vuong, June Goto, Rolf Stottmann, Kenneth Campbell, and Francesco Mangano
Hydrocephalus is the most common brain malformation found at birth. Although the surgical intervention can greatly ameliorate outcomes, currently there is no medical cure for this condition. In addition, about 30% of these cases have unknown etiology. In order to identify molecular mechanisms involved in congenital hydrocephalus development, we investigated the genetic mutation responsible for progressive hydrocephaly (prh) mouse mutant, which was isolated in a previous forward genetic screening for severe neonatal onset hydrocephalus phenotype in mice. We performed a whole-genome sequencing in the mutant mouse and found a single nucleotide mutation candidate within Ccdc39 (coiled-coil domain containing protein 39) gene, one of primary ciliary dyskinesia genes critical for motile cilia functions. Western blotting and cDNA sequencing analysis show that the mutation affects proper mRNA splicing of the Ccdc39 gene and results in loss of the protein to undetectable levels. In immunohistochemistry, we found Ccdc39 is highly expressed in choroid plexus epithelium cells in the developing wild type mouse brain, but is missing from that of prh mutant. Choroid plexus is the major cerebrospinal fluid production site and has transiently motile multi-cilia in neonatal period. The transmission electron microscopy study revealed microtubule structures of choroid plexus cilia axoneme is disrupted in the prh mutant mice. In vitro trancytosis assay using primary cultured mouse choroid plexus cells showed altered fluid transferring rate in the mutant derived cells. Together, these data indicate that loss of Ccdc39 may disrupt the motility of choroid plexus cilia in the developing brain and suggest the possible involvement of choroid plexus cilia in the development of congenital hydrocephalus.
Congenital hydrocephalus continues to be a difficult disease to treat. Unfortunately research which explains the exact mechanisms leading to the development of this disease is lacking, likely as a result of no robust models.
In 2011, Stottmann et al, were looking to find and understand the genes involved with neurodevelopment. In a mouse model, their lab performed a forward genetic screen using ENU to produce novel mutations with the goal of modeling human genetic defects. During the screen, they produced a mutation which they called “progressive hydrocephalus” (prh). At birth, these mice appear normal, but at day 14 they are visibly hydrocephalic and do not survive to the weaning period. The image above shows how the mice look compared to wild type (wt) mice.
Recently our lab discovered a gene that is completely devoid in the prh mice called coiled-coil domain containing protein 39 (Ccdc39). This gene was found to be richly expressed in cells containing cilia and was ultimately found to be required for the assembly of inner dynein arms for the normal ciliary motility in humans and dogs (Merveille et al.). This is important because according to Lodish et al.:
the inner-arm dyneins are responsible for producing the sliding forces that are converted to bending; this suggests that inner-arm dyneins are essential for bending
Essentially, if the inner dynein of cilia are not assembled correctly, bending forces within the cilia cannot be generated which then would greatly affect motility of the cilia and thus it’s main function.
Our lab has preliminary data that suggests the prh mutation results in loss of ccdc39 protein within the choroid plexus, and this is what may be causing the hydrocephalus phenotype seen in prh mice. Thus aims of our research include:
- Proving the ccdc39 mutation in prh mice is the cause of the hydrocephalus
- Then selectively knocking out the ccdc39 gene in the choroid plexus to prove that cilia disruption within the choroid plexus itself is responsible for the hydrocephalus phenotype seen in prh mice
- Show that CSF production is abnormal in choroid plexus cells of the ccdc39 mutants
I hope to gather enough data this year to accomplish each of these goals. I am excited for this year in the lab and am thankful to Dr. Mangano and Dr. Goto for their mentorship.
Stottmann, R. W., Moran, J. L., Turbe-Doan, A., Driver, E., Kelley, M., & Beier, D. R. (2011). Focusing forward genetics: a tripartite ENU screen for neurodevelopmental mutations in the mouse. Genetics, 188(3), 615–624. http://doi.org/10.1534/genetics.111.126862
Lodish H, Berk A, Zipursky SL, et al. Molecular Cell Biology. 4th edition. New York: W. H. Freeman; 2000. Section 19.4, Cilia and Flagella: Structure and Movement. Available from: http://www.ncbi.nlm.nih.gov/books/NBK21698/
Merveille, A.-C., Davis, E. E., Becker-Heck, A., Legendre, M., Amirav, I., Bataille, G., et al. (2011). CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs. Nature Genetics, 43(1), 72–78. http://doi.org/10.1038/ng.726
New evidence by the Cochrane Collaboration shows that spinal manipulation (or adjustments) are no better at treating acute (6 weeks or less) lower back pain, than over the counter ibuprofen or other NSAIDS. In fact, the back pain resolves on its own in most cases. Here is what the Cochrane Collaboration had to say about their findings:
Low-back pain is a common and disabling disorder, representing a great burden both to the individual and society. It often results in reduced quality of life, time lost from work, and substantial medical expense. Spinal manipulative therapy (SMT) is widely practised by a variety of healthcare professionals worldwide and is a common choice for the treatment of low-back pain. The effectiveness of this form of therapy for the management of acute low-back pain is, however, not without dispute.
For this review, acute low-back pain was defined as pain lasting less than six weeks. Only cases of low-back pain not caused by a known underlying condition, for example, infection, tumour, or fracture, were included. Also included were patients whose pain was predominantly in the lower back but may also have radiated (spread) into the buttocks and legs.
SMT is known as a ‘hands-on’ treatment directed towards the spine, which includes both manipulation and mobilization. The therapist applies manual mobilization by passively moving the spinal joints within the patient’s range of motion using slow, passive movements, beginning with a small range and gradually increasing to a larger range of motion. Manipulation is a passive technique whereby the therapist applies a specifically directed manual impulse, or thrust, to a joint at or near the end of the passive (or physiological) range of motion. This is often accompanied by an audible ‘crack’.
In this review, a total of 20 randomized controlled trials (RCTs) (representing 2674 participants) assessing the effects of SMT in patients with acute low-back pain were identified. Treatment was delivered by a variety of practitioners, including chiropractors, manual therapists, and osteopaths. Approximately one-third of the trials were considered to be of high methodological quality, meaning these studies provided a high level of confidence in the outcome of SMT.
Overall, we found generally low to very low quality evidence suggesting that SMT is no more effective in the treatment of patients with acute low-back pain than inert interventions, sham (or fake) SMT, or when added to another treatment such as standard medical care. SMT also appears to be no more effective than other recommended therapies. SMT appears to be safe when compared to other treatment options but other considerations include costs of care.