| dc.description.abstracteng | The MECP2 gene is located in the Xq28, which encodes for methyl-CpG binding protein 2 (MeCP2). Mutations in MECP2 gene is the primary cause of postnatal neurodevelopmental disorder, Rett syndrome (RTT) (loss-of-function). In contrast the over expression of MECP2 leads to duplication syndrome (gain-of-function). In both cases, gain and loss-of function of MECP2 leads to neurological disorders.
In the first part of the thesis, we attempted to characterize the pathomechanism associated with human R270X mutation in MECP2, which is one of the most frequent mutation in RTT cohorts. The R270X mutation is located within the transcriptional repression domain- nuclear localization signals (TRD-NLS) of MeCP2 protein. This nonsense mutation exhibits severe phenotype with higher mortality rate as compared to the other mutations in RTT cohorts. To evaluate the molecular and functional role of R270X mutation, we generated a Mecp2R270_EGFP transgenic mouse by BAC recombineering technology, which is equivalent to human R270X mutation. The Mecp2R270_EGFP transgenic mouse showed similar pattern of expression as compared to the endogenous MeCP2. Further, we bred the Mecp2R270_EGFP transgenic male mouse with Mecp2 knockout female to obtain Mecp2R270 knockin (KI) mouse. Phenotypical evaluation revealed no neurological impairment in Mecp2R270_EGFP transgenic mice, but KI mice were positive for hindlimb clasping. In addition, we observed no difference in neurite length, area of soma and spine density between wildtype and Mecp2R270_EGFP neurons, but detected reduced neurite length, soma size and spine number in KI neurons. From our experimental data, we conclude that MeCP2R270_EGFP protein has a deleterious effect on neuronal morphology and alters the spine density.
In the second part of the thesis, we generated a Mecp2 transgenic mouse model to determine the MECP2 dosage effect on duplication syndrome. The generated Mecp2WT_EGFP transgenic mice mildly overexpressed MeCP2 (~1.5X) (MeCP2 transgenic with endogenous) From our extensive behavioral analyzes, we observed increased aggressiveness and higher seizures in Mecp2WT_EGFP transgenic mice propensity after treatment with epileptogenic compound pentylenetetrazole (PTZ). Furthermore, induction of Mecp2WT_EGFP transgenic cultured neurons with PTZ caused increase calcium amplitude with a higher frequency. Additionally, the ex vivo and in vivo evaluation of neuronal parameters of hippocampal neurons revealed an increased area of soma, reduced tertiary branching sites and increased spine density in Mecp2WT_EGFP transgenic mouse. Collectively, our results suggest that mild MeCP2 overexpression in mice leads to epileptic seizures as a first symptom. Furthermore, precise MeCP2 dosage is necessary for proper neurodevelopment in mice.
In the third part of the thesis, we reported that the MeCP2 expression is not restricted only in the neurons but also expressed in astrocytes. Recent studies highlighted the role of MeCP2 in astrocytes. The co-cultured assay system with Mecp2 null astrocytes together with wild type neurons displayed shorter and small number of dendrites. These findings suggested the unknown secreted factor(s) from Mecp2 deficient astrocytes cause toxic effect to the neighboring neurons in non-cell-autonomous manner. In order to determine and characterize the MeCP2 targets in astrocytes, we performed proteomic analysis and characterized ~5000 different proteins in astrocytes with three independent technical repeats. Our proteomic analysis revealed 69 up-regulated and 29 down-regulated proteins in knockin (KI) and 50 up-regulated and 32 down-regulated proteins in knockout (KO), when compared to wildtype astocytes. The identified Mecp2 target proteins from proteomic analysis revealed that MeCP2 is involved in cell adhesions through desmosomal proteins (Dsp and Pkp1), regulation of reactive oxygen species (ROS) (Txn2) and glutamate homeostasis via Slc25a18 in astrocytes. We could validate the differential expression of Mecp2 target proteins such as Dsp and Pkp1 in Mecp2 knockout astrocytes compared to wild type astrocytes through Western blot analysis. After validating the proteomic data, in future an ex vivo assay can be established to test the effect of non-cell autonomous astrocytes factors identified through proteomics approach. Establishing the role of glial dysfunction in RTT pathogenesis could provide a new avenue for understanding RTT and will assist us in developing novel therapy for this disease. | de |