# Upregulation of Neuronal Rheb(S16H) for Hippocampal Protection in the Adult Brain
## 5 Mutation of Rheb Leading to Constitutive Activation  
The expression levels of growth-associated proteins are lower in adulthood than in the process of development. Consequently, the regenerative capacity of mature neurons is limited in the aging brain [127]. As described previously, Rheb-mTOR signaling is a key regulator of various mechanisms associated with neuronal survival and regeneration. Accordingly, several studies have identified Rheb mutations that lead to constitutive activation (Table 1). The mutation of serine to histidine at position 16 of Rheb [Rheb(S16H)] has been found to exhibit gain-of-function properties, resulting in highly activated mTOR signaling in TSC1/2-overexpressing cells [128]. Our previous studies showed that the overexpression of Rheb(S16H) could significantly increase the levels of NTFs such as GDNF, CNTF, and BDNF in the adult brain [5; 11; 26; 29; 69]. The mutation of tyrosine to asparagine at position 35 of Rheb [Rheb(Y35N)] in renal cell carcinoma has been reported to cause mTOR1 hyperactivation by increasing the resistance to TSC2 GAP activity, resulting in the hyperproliferation of tumor cells [129]. Another study identified the mutation of glycine to alanine at position 63 of Rheb [Rheb(G35A)], which could stimulate the phosphorylation of S6 kinase more strongly compared wild-type Rheb in HeLa cells [130]. The mutation of glutamine to leucine at position 64 of Rheb [Rheb(Q64L)] was found to strongly induce oncogenic transformation in chicken embryonic fibroblast cultures. Cells with Rheb(Q64L) overexpression could promote the constitutive phosphorylation of the ribosomal proteins S6K and 4E-BP1 [131] and exhibit increased GTP loading and partial resistance to TSC-GAP [132]. Another study identified specific Rheb hyperactive mutations in four different residues: (a) V17G and V17A, (b) S21G and S21I, (c) K120R, (d) N153S and N153T [133]. In comparison with wild-type Rheb, these hyperactive mutations of Rheb conferred resistance to canavanine toxicity. The GTP-binding affinity of these mutations was closely similar to that of wild-type Rheb, whereas the GDP-binding affinity was greatly reduced. In our previous study, we demonstrated the neuroprotective potential of the constitutively active forms of Rheb with mutations such as Rheb(S16H) and Rheb(N153T) in a neurotoxin-induced animal model of PD [24]. In particular, Rheb(S16H) upregulation by adeno-associated virus serotype 1 (AAV1) administration in nigral dopaminergic neurons significantly increased the activation of mTOR1, resulting in greater neuroprotective effects compared with Rheb(N153T) upregulation in the animal model of PD [24]. In addition, our studies demonstrated that the upregulation of neuronal Rheb(S16H) could protect neurons against neurodegeneration and promote axonal regrowth in the nigrostriatal dopamine system and hippocampus of the adult brain [11; 24; 25; 30; 69; 134]. The activation of mTOR1 induced by Rheb(S16H) transduction of hippocampal neurons was found to result in BDNF production, which protected against thrombin-induced neurotoxicity in the rat hippocampus [5]. In the same manner, AAV1-Rheb(S16H) transduction in the hippocampus of transgenic AD mice prevented cognitive function impairment [26; 29].  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Amino Acid Position** & **Mutation** & **Cell Type** & **Reference** \\ \hline
**16** & S16H & TSC1/2-overexpressing HEK293T & [128] \\ \hline
**17** & V17G, V17A & fission yeast & [133] \\ \hline
**21** & S21G and S21I & fission yeast & [133] \\ \hline
**35** & Y35N & renal cell carcinoma & [129] \\ \hline
**63** & G63A & HeLa cells & [130] \\ \hline
**64** & Q64L & chicken embryonic fibroblasts & [131] \\ \hline
**120** & K120R & fission yeast & [133] \\ \hline
**153** & N153T & fission yeast & [128; 133] \\ \cline{3-