To address this question, we used an in vivo model of tau pathology based on intracerebroventricular injections of AAV2/6 vectors in mouse neonates, to induce widespread overexpression of various forms of human tau in the forebrain. Tau pathology developed in the cortex and hippocampus, and induced motor behavioral deficits. To test the hypothesis that the propensity of the microtubule-binding domain to form β-sheets is a key factor in tau toxicity, we compared three variants of human 4R0N tau. The wild-type (WT) form was first compared to the pathogenic P301S mutant linked to FTDP-17. Conversely, to reduce the propensity of the microtubule-binding domain to form β-sheets, we introduced β-sheet-breaking proline residues in WT tau. The results show that the neuronal dysfunction caused by tau overexpression depends on the propensity of the microtubule-binding domain to form β-sheets. In addition, the observed behavioral defects correlate with differences in tau hyperphosphorylation and aggregation, as well as with the propensity of tau to protect neurites exposed to the microtubule-destabilizing agent vinblastine. These findings indicate that amino acid substitutions, or other modifications that may affect tau conformation in the microtubule-binding repeats, determine the progression and the type of tau-induced pathology in neuronal cells in vivo.