Consejo de Etnias Colegio San Bernardino IED

5.4.1 TRNP1 expression in the developing human brain

Down regulation of Trnp1 resulted in increased numbers of BPs and oRGs with a “fanned array” expansion of the cortex, which ultimately led to folding and pseudo-gyrification of the otherwise lissencephalic murine brain. These observations initiated the hypothesis that

Trnp1 may be an important regulator of higher mammalian brain development. It has been suggested, that gyri and sulci in gyrencephalic brains form due to differences in progenitor expansions (Smart and McSherry, 1986a; 1986b; Kriegstein et al., 2006). In this scenario local differences of Trnp1 expression levels could regulate differential expansion in folded brains. To adress this question, our collaborator Victor Borell was investigating TRNP1

expression in the human developing brain. In situ mRNA analysis demonstrated the expression of TRNP1 in the developing human brain. Remarkably, TRNP1 is expressed in a similar manner in the human brain as it is in the murine brain with high levels of TRNP1

in the ventricular zone and in newborn neurons in the cortical plate, but rather low levels in the subventricular zone where the basal progenitors and outer radial glial cells are located in the human brain. However, in strong contrast to the murine brain local differences of

TRNP1 expression within the VZ were clearly detectable in the human brain. Supporting the role of TRNP1 in cortical expansion, low levels of TRNP1 in the VZ were associated with a rather expanded cortical area, whereas high levels of TRNP1 in the VZ correlated with less neurons in the CP. This expression pattern is intriguing in regard to my observation in the murine brain in vivo where manipulation of the protein resulted in differences of radial glial cell fate with high levels of Trnp1 leading to self renewal of RGs and tangential expansion, whereas low levels of Trnp1 provoked amplification of neuronal output and radial expansion through production of basal progenitors and outer radial glial cells. The local differences of TRNP1 expression in the VZ of the human brain may therefore be causative for folding and differential expansion of different regions within the human brain resulting in its gyrencephaly. This is in agreement with previously suggested models in which a locally enlarged oSVZ correlated with increased expansion (Smart and McSherry, 1986a; 1986b; Kriegstein et al., 2006; Martínez-Cerdeño et al., 2012).

Figure 39: TRNP1 expression in the developing human brain

In situ analysis of TRNP1 transcript expression in the human brain. Embryonic brain sections of gestation week 12 and 21 were hybridized with TRNP1 antisense probe. (A-A’’) In situ hybridization on embryonic sections of gestation week 12. (B-B’’) in situ hybridization on embryonic sections of gestation week 21. Note the differential expression of TRNP1 in the VZ of different areas with high levels of TRNP1 in the VZ correlating with low numbers of neurons in the CP of the same region and low-level expression of TRNP1 in the VZ correlating with increased numbers of neurons in the CP.

5.4.2 Investigation of a possible association of TRNP1 with human diseases

The presence of TRNP1 in the human brain emphasized the significance of the findings of this work and also led to the question whether TRNP1 may also be disease associated. Especially diseases with developmental neurogenesis defects such as lissencephaly or mental retardations could be possibly caused by genetic mutations of TRNP1. Online search using well established databases such as the genetic association database (http://geneticassociationdb.nih.gov/) the human gene mutation database (http://www.hgmd.cf.ac.uk) for human disease cases associated with deletions or mutations

of TRNP1 did not reveal a single case of TRNP1 affected individuals. However, personal internet search revealed one case of a genomic duplication including TRNP1. This particular case carries a duplication comprising 763 kilobases of chromosome 1p36.11 including a duplication of TRNP1 (position: chr. 1 26716140 - 27480136; see Figure 40). Other than this genetic duplication no genetic abnormalities were found using 244k- Oligonucleotide Array (Agilent) and the duplication does not represent a known Polymorphism. The above female patient is now six years old and was diagnosed with Rett syndrome. The typical “washing movements” initially gave a first hint towards the syndrome causative for her disability. In contrast to this patient that carries a duplication on chromosome 1, Rett syndrome is typically an X-chromosomal dominantly inherited disease. Male fetuses with the disorder rarely survive to term. The main genetic background of Rett syndrome is mutations in the gene MeCP2 that is encoded on the X- chromosome. Loss of function mutations in MeCP2 can arise sporadically or within the germ line and are associated with 75% of Rett cases (Hoffbuhr et al., 2001; 2002). However, in less than 10% of Rett cases, mutations in the genes CDKL5 or FOXG1 (involved in control of proliferation of apical progenitors in forebrain development (Manuel et al., 2011)) have also been found to cause the syndrome (Tao et al., 2004; Weaving et al., 2004; 2005; Chen et al., 2010). Today, Rett syndrome is still diagnosed by clinical observation, and in some very rare cases, no known mutated gene can be found. These clinical observations prompt the hypothesis that Trnp1 might be directly or indirectly associated with MeCP2 thereby causing a similar phenotype as MeCP2 mutations. It is conceivable, that Trnp1 may represent a downstream target, a co-factor or a competitor of MeCP2 and further investigations need to be performed in order to elucidate this association. The duplicated chromosomal region of course also includes other genes and an effect of the combined action of more than only one gene that is duplicated is possible. Nevertheless, the importance of Trnp1 during cortical development together with the severe syndrome observed in a case of chromosomal duplication in humans indicates, that Trnp1 may also play a central role in development of the human brain.

Figure 40: Chromosomal duplication of a girl presented with Rett syndrome

Graphical representation of human chromosome 1 - region 1p36.11. The region between 26 598 139 and 27 598 138 is depicted. The region duplicated in the female patient with Rett syndrome is marked with a red square. Genes are labeled as indicated. The TRNP1 gene is underlined in red.

Additionally two cases of chromosomal abnormalities in a region similar to the mentioned case (also comprising the TRNP1 gene locus) have recently been reported on the DECIPHER homepage (https://decipher.sanger.ac.uk). Both patients showed mental retardation. One female patient carries a deletion comprising 1.97 megabases (chr. 1 27237958 - 29205507) with 38 genes affected (including TRNP1). The other diseased case, a male patient with unknown age has two chromosomal regions affected: a deletion on chromosome 6 comprising 1.11 megabases (chr.6 169786414 - 170892243) with 10 genes affected and a duplication on chromosome 1 comprising 580 kilobases (chr. 1 27190888 – 27773330) with 17 genes (including TRNP1) affected. Remarkably, the overlap of all the three cases comprises a very small region of only 242 kilobases on chromosome 1 including only 6 genes: NUDC, NR0B2, C1orf172, TRNP1, FAM46B and SLC9A1

(27237958 – 27480136). Additionally, the latter patient also presented with lissencephaly (possibly due to increased TRNP1-levels as only timed loss of the protein was able to induce radial expansion and folding of the otherwise lissencephalic murine brain). However, in the latter case another gene known to play a central role in neurogenesis was affected: DLL1 (deletion on chr. 6).

While some hypotheses emerge in regard to the cellular and possibly disease associated function of Trnp1, it is important to also understand how it may exert its fascinating roles at the molecular level.