Cardiofaciocutaneous Syndrome-Associated BRAF Mutants in Development and Cancer

Cardiofaciocutaneous syndrome (CFCS) is an autosomal-dominant disorder caused by germ-line mutations in members of the RAS/ERK signaling pathway, most commonly in BRAF. Selected mutations that either activate or impair BRAF kinase activity and that are known to cause CFCS have also been identified in melanoma. I found that knock-in mouse mutants expressing BrafK499E (kinase-activating) and BrafG469E (kinase-impaired) alleles have many features of CFCS, including short stature, craniofacial dysmorphia, and an increased heart weight to body weight ratio. Studies of the phenotype of BrafK499E mice show that the effects of this mutation might be affected by genetic modifiers on the 129/B6 mouse background. Endogenous expression of BrafG469E in mouse embryonic fibroblasts (MEFs) caused increased MEK and ERK activation compared to wild-type MEFs . These effects correlated with increased heterodimerization of mutant BRAF with RAF1, as assessed by co-immunoprecipitation. Finally, I found that BrafK499E expression induced post-natally in vivo in melanocytes is not sufficient to cause hyperpigmentation or nevus formation, unlike the highly activated melanoma-associated allele BrafV600E .M.Sc

Lafora disease

Lafora disease (LD) is an autosomal recessive progressive myoclonus epilepsy due to mutations in the EPM2A (laforin) and EPM2B (malin) genes, with no substantial genotype-phenotype differences between the two. Founder effects and recurrent mutations are common, and mostly isolated to specific ethnic groups and/or geographical locations. Pathologically, LD is characterized by distinctive polyglucosans, which are formations of abnormal glycogen. Polyglucosans, or Lafora bodies (LB) are typically found in the brain, periportal hepatocytes of the liver, skeletal and cardiac myocytes, and in the eccrine duct and apocrine myoepithelial cells of sweat glands. Mouse models of the disease and other naturally occurring animal models have similar pathology and phenotype. Hypotheses of LB formation remain controversial, with compelling evidence and caveats for each hypothesis. However, it is clear that the laforin and malin functions regulating glycogen structure are key. With the exception of a few missense mutations LD is clinically homogeneous, with onset in adolescence. Symptoms begin with seizures, and neurological decline follows soon after. The disease course is progressive and fatal, with death occurring within 10 years of onset. Antiepileptic drugs are mostly non-effective, with none having a major influence on the progression of cognitive and behavioral symptoms. Diagnosis and genetic counseling are important aspects of LD, and social support is essential in disease management. Future therapeutics for LD will revolve around the pathogenesics of the disease. Currently, efforts at identifying compounds or approaches to reduce brain glycogen synthesis appear to be highly promising

PTG depletion removes Lafora bodies and rescues the fatal epilepsy of Lafora disease.

Lafora disease is the most common teenage-onset neurodegenerative disease, the main teenage-onset form of progressive myoclonus epilepsy (PME), and one of the severest epilepsies. Pathologically, a starch-like compound, polyglucosan, accumulates in neuronal cell bodies and overtakes neuronal small processes, mainly dendrites. Polyglucosan formation is catalyzed by glycogen synthase, which is activated through dephosphorylation by glycogen-associated protein phosphatase-1 (PP1). Here we remove PTG, one of the proteins that target PP1 to glycogen, from mice with Lafora disease. This results in near-complete disappearance of polyglucosans and in resolution of neurodegeneration and myoclonic epilepsy. This work discloses an entryway to treating this fatal epilepsy and potentially other glycogen storage diseases

LB in brain of 12 month-old LKO and DKO mice.

<p>(a–b) Frontal cortex and hippocampus respectively from a LKO mouse stained with PAS-D. Note abundant LB within the neuropil and in the perikarya of numerous neurons. (c–d) Comparable regions from a DKO mouse. Arrows, examples of LB. All bars, 50 µm.</p

Glycogen levels in skeletal muscle and brain in 12-month-old wt, LKO, and DKO mice (µmol glucose/gm tissue).

<p>Skeletal muscle (a) and brain (b).</p

LB in skeletal muscle.

<p>(a) Muscle from a LKO mouse stained with PAS-D. Note presence of numerous LB in many fibers; bar, 100 µm; arrows, LB-replete myofibers; arrowheads, myofibers not containing LB. (b) Comparable field from a DKO mouse; bar, 50 µm. Higher magnification chosen for the DKO example to illustrate lack of even small LB.</p

Gliosis in LKO mice.

<p>(a) Hippocampus of a LKO mouse stained with GFAP. Note the large numbers of GFAP-positive astrocytes. (b) Comparable region from a DKO mouse. Bars, 100 µm. Arrows, astrocytes; arrowheads, gliosis. (c) Counts of GFAP-positive astrocytes. For significance, whole brain p<0.02; hippocampus p<0.001; cerebellum, not significant; frontal cortex, p<0.002 (ANOVA); n = 4–7 per genotype.</p

LB numbers in brain.

<p>(a) Morphometric analysis of granular LB in whole brain and different brain regions. Granular is the histochemical description of the small LB in the neuropil, which by electron microscopy are shown to be in neuronal processes, mainly dendrites. Statistics: p<0.001 in all regions between LKO and DKO (ANOVA); n = 4 per genotype. (b) Morphometric analysis of perikaryal LB. Statistics: p<0.001 between LKO and DKO, except in cerebellum, where the difference is not significant (ANOVA); n = 4 per genotype.</p

Myoclonus per minute in wt, DKO, and LKO mice.

<p>n = nine to 12 mice per genotype.</p