Forebrain depletion of Rheb GTPase elicits spatial memory deficits in mice

Abstract

The precise molecular and cellular events responsible for age-dependent cognitive dysfunctions remain unclear. We report that Rheb (ras homolog enriched in brain) GTPase, an activator of mammalian target of rapamycin (mTOR), regulates memory functions in mice. Conditional depletion of Rheb selectively in the forebrain of mice obtained from crossing Rheb^f/f and CamKII^Cre results in spontaneous signs of age-related memory loss, that is, spatial memory deficits (T-maze, Morris water maze) without affecting locomotor (open-field test), anxiety-like (elevated plus maze), or contextual fear conditioning functions. Partial depletion of Rheb in forebrain was sufficient to elicit memory defects with little effect on the neuronal size, cortical thickness, or mammalian target of rapamycin activity. Rheb depletion, however, increased the levels of beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), a protein elevated in aging and Alzheimer's disease. Overall, our study demonstrates that forebrain Rheb promotes aging-associated cognitive defects. Thus, molecular understanding of Rheb pathway in brain may provide new therapeutic targets for aging and/or Alzheimer's disease–associated memory deficits.

Introduction

Aging is a major risk factor for cognitive decline and dementia. Dissecting the age-related mechanisms becomes important, not only because it is a fundamental biological question that we do not yet understand completely but also because memory affecting disorders, such as Alzheimer disease (AD) and related dementia, are a major problem that affect the elderly population. What age-related factors determine the age of onset, and how the age of onset relates to the selective dysfunction of neurons in the brain, remains less understood. Rheb (Ras homolog enriched in brain) belongs to Ras family of GTPase highly enriched in brain and is induced in the brain in an activity-dependent manner (Yamagata et al., 1994). Classically, Rheb GTPase is implicated in the activation of mammalian target of rapamycin (mTOR), a kinase with diverse cellular functions. mTOR signaling has been implicated in life-span extension (Johnson et al., 2013), as well as in behavioral and pathological symptoms in neurodegenerative diseases (Korolchuk et al., 2010, Ravikumar et al., 2004), including AD (Caccamo et al., 2010, Spilman et al., 2010). mTOR pathway also intersects with the degradation pathway, autophagy, which is another important modulator of aging (Rubinsztein et al., 2011). For example, pharmacological inhibition of mTOR (e.g., with rapamycin), genetic manipulations of genes (PI3Ka, ATG-7, mTOR) or calorie restriction that increase life span in model organisms often stimulate autophagy, and its inhibition compromises the longevity-promoting effects (Inuzuka et al., 2009, Juhasz et al., 2007, Lamming et al., 2012, Onal et al., 2013, Rubinsztein et al., 2011, Ruckenstuhl et al., 2014). A direct role for Rheb in aging was also demonstrated in Caenorhabditis elegans (Honjoh et al., 2009). But the molecular mechanisms of how Rheb GTPase participates in aging and neurodegenerative disorders remain unclear.

Earlier we found a biochemical link for Rheb and beta-site amyloid precursor protein cleaving enzyme 1 (BACE1), a critical enzyme that generates Aβ (Shahani et al., 2014), which is increased in normal aging, sporadic AD, familial AD, and in mild cognitive impairment (Cheng et al., 2014, Kandalepas and Vassar, 2012, Kuhn et al., 2012, Sinha et al., 1999, Vassar et al., 1999, Vassar and Kandalepas, 2011). We reported that Rheb regulates the BACE1 protein levels; while overexpression of Rheb reduces BACE1 in the intact hippocampus, Rheb depletion enhances BACE1 protein levels in cultured cortical neurons (Shahani et al., 2014). The physiological implication of the Rheb–BACE1 link in brain function remains unknown. Homozygous deletion of Rheb was embryonically lethal, hindering the study of Rheb functions in adult brain. Heterozygous Rheb mice, however, do breed and have normal life spans. A recent study in which a further depletion of Rheb using an inducible promoter in the forebrain (Rheb^f/−; CaMKIIα-Cre^ERT2) found no behavioral or anatomical defects in mice that were of 2–5 months old (Goorden et al., 2015). In another study where Rheb was depleted in neural progenitor cells by Nes-cre (Rheb1^f/f; Nes-Cre), there were no apparent defects in the development of progenitor cells, but most of Rheb1^f/f; Nes-Cre mice died at ∼5–6 weeks old with severe defects in myelination (Zou et al., 2011).

To further assess the role of Rheb in brain functions, we crossed Rheb^f/f mice with transgenic mice (CaMKIIα) that constitutively express Cre in the forebrain beginning after P5 (Dragatsis and Zeitlin, 2000), and we report motor, cognitive, biochemical, and morphological alterations upon forebrain Rheb depletion in mice that were 6–8 months old.