Protease activated receptor 2 controls myelin development, resiliency and repair.

Oligodendrocytes are essential regulators of axonal energy homeostasis and electrical conduction and emerging target cells for restoration of neurological function. Here we investigate the role of protease activated receptor 2 (PAR2), a unique protease activated G protein-coupled receptor, in myelin development and repair using the spinal cord as a model. Results demonstrate that genetic deletion of PAR2 accelerates myelin production, including higher proteolipid protein (PLP) levels in the spinal cord at birth and higher levels of myelin basic protein and thickened myelin sheaths in adulthood. Enhancements in spinal cord myelin with PAR2 loss-of-function were accompanied by increased numbers of Olig2- and CC1-positive oligodendrocytes, as well as in levels of cyclic adenosine monophosphate (cAMP), and extracellular signal related kinase 1/2 (ERK1/2) signaling. Parallel promyelinating effects were observed after blocking PAR2 expression in purified oligodendrocyte cultures, whereas inhibiting adenylate cyclase reversed these effects. Conversely, PAR2 activation reduced PLP expression and this effect was prevented by brain derived neurotrophic factor (BDNF), a promyelinating growth factor that signals through cAMP. PAR2 knockout mice also showed improved myelin resiliency after traumatic spinal cord injury and an accelerated pattern of myelin regeneration after focal demyelination. These findings suggest that PAR2 is an important controller of myelin production and regeneration, both in the developing and adult spinal cord.

A dominantly acting murine allele of Mcm4 causes chromosomal abnormalities and promotes tumorigenesis.

Here we report the isolation of a murine model for heritable T cell lymphoblastic leukemia/lymphoma (T-ALL) called Spontaneous dominant leukemia (Sdl). Sdl heterozygous mice develop disease with a short latency and high penetrance, while mice homozygous for the mutation die early during embryonic development. Sdl mice exhibit an increase in the frequency of micronucleated reticulocytes, and T-ALLs from Sdl mice harbor small amplifications and deletions, including activating deletions at the Notch1 locus. Using exome sequencing it was determined that Sdl mice harbor a spontaneously acquired mutation in Mcm4 (Mcm4(D573H)). MCM4 is part of the heterohexameric complex of MCM2-7 that is important for licensing of DNA origins prior to S phase and also serves as the core of the replicative helicase that unwinds DNA at replication forks. Previous studies in murine models have discovered that genetic reductions of MCM complex levels promote tumor formation by causing genomic instability. However, Sdl mice possess normal levels of Mcms, and there is no evidence for loss-of-heterozygosity at the Mcm4 locus in Sdl leukemias. Studies in Saccharomyces cerevisiae indicate that the Sdl mutation produces a biologically inactive helicase. Together, these data support a model in which chromosomal abnormalities in Sdl mice result from the ability of MCM4(D573H) to incorporate into MCM complexes and render them inactive. Our studies indicate that dominantly acting alleles of MCMs can be compatible with viability but have dramatic oncogenic consequences by causing chromosomal abnormalities.

Astrocyte heterogeneity across the brain and spinal cord occurs developmentally, in adulthood and in response to demyelination.

Astrocytes have emerged as essential regulators of function and response to injury in the brain and spinal cord, yet very little is known about regional differences that exist. Here we compare the expression of key astroglial markers (glial fibrillary acidic protein (GFAP) and Aldehyde Dehydrogenase-1 Family Member L1 (ALDH1L1)) across these disparate poles of the neuraxis, tracking their expression developmentally and in the context of demyelination. In addition, we document changes in the astrocyte regulatory cytokine interleukin 6 (IL-6), and its signaling partner signal transducer and activator of transcription 3 (STAT3), in vivo and in vitro. Results demonstrate that GFAP expression is higher in the developing and adult spinal cord relative to brain. Comparisons between GFAP and ALDH1L1 expression suggest elevations in spinal cord GFAP during the early postnatal period reflect an accelerated appearance of astrocytes, while elevations in adulthood reflect higher expression by individual astrocytes. Notably, increases in spinal cord compared to whole brain GFAP were paralleled by higher levels of IL-6 and STAT3. Equivalent elevations in GFAP, GFAP/ALDH1L1 ratios, and in IL-6, were observed in primary astrocyte cultures derived from spinal cord compared to cortex. Also, higher levels of GFAP were observed in the spinal cord compared to the brain after focal demyelinating injury. Altogether, these studies point to key differences in astrocyte abundance and the expression of GFAP and IL-6 across the brain and spinal cord that are positioned to influence regional specialization developmentally and responses occurring in the context of injury and disease.

Clinical significance and novel mechanism of action of kallikrein 6 in glioblastoma.

BACKGROUND: Kallikreins have prognostic value in specific malignancies, but few studies have addressed their clinical significance to glioblastoma multiforme (GBM). Kallikrein 6 (KLK6) is of potential high relevance to GBM, since it is upregulated at sites of CNS pathology and linked to reactive astrogliosis. Here we examine the clinical value of KLK6 as a prognostic indicator of GBM patient survival and its activity in promoting resistance to cytotoxic agents. METHODS: The association between patient survival and levels of KLK6 immunoreactivity were investigated in 60 grade IV astrocytoma tumor specimens. Levels of KLK6 RNA were also evaluated in a separate set of GBM patient tumors (n = 23). Recombinant KLK6 or enforced KLK6 overexpression in GBM cell lines was used to evaluate effects on astrocytoma cell survival. RESULTS: A range of KLK6 expression was observed across grade IV tumors, with higher levels a poor prognostic indicator of patient survival (P = .02) even after adjusting for gender and Eastern Cooperative Oncology Group performance scores (P = .01). KLK6 reduced the sensitivity of GBM cell lines to cytotoxic agents, including staurosporine and cisplatin, and to the current standard of patient care: radiotherapy or temozolomide alone or in combination. The ability of KLK6 to promote resistance to apoptosis was dependent on activation of the thrombin receptor, protease activated receptor 1. CONCLUSIONS: Taken together, these results indicate that elevated levels of KLK6 in GBM are likely to promote the resistance of tumor cells to cytotoxic agents and are an indicator of reduced patient postsurgical survival times.