Although apoE4 and impaired lipid metabolism have been implicated in the pathogenesis of AD, the underlying molecular mechanisms and pathways are not clear. In this study, we showed that deletion of the apoE/lipoprotein receptor LRP1 from forebrain neurons of adult mice leads to global impairment in brain lipid metabolism. More interestingly, the reduced brain lipid levels correlated with progressive, age-dependent degeneration of dendritic spines and synapses, impaired neurobehaviors, and eventual neurodegeneration. We further showed that reduced levels of selective glutamate receptors may contribute to impaired synaptic functions in LRP1-KO mice. Together, our work provides strong evidence that brain lipid metabolism is critical for maintenance of the integrity and functions of neuronal synapses, and establishes a novel mouse model in which impaired brain lipid metabolism leads to age-dependent dendritic spine, synapse and neurodegeneration.
LRP1 is a major neuronal receptor for brain apoE/lipoprotein (Liu et al., 2007
; Bu, 2009
). It is highly expressed in neurons and its rapid endocytosis (Li et al, 2000
) and efficient recycling (Van Kerkhof et al., 2005
) likely contribute to its role as a key lipid transporter for neurons. Cholesterol is an essential component of membranes and myelin sheaths and is crucial for synaptic integrity and neuronal function. Because LRP1 levels are significantly reduced in AD brains, which correlate with increased AD susceptibility (Kang et al., 2000
), it is tempting to speculate that decreased LRP1 levels in AD could be directly responsible for cholesterol loss and related synaptic dysfunction independently of Aβ accumulation. GalCer and its sulfated derivative, sulfatide, are major lipid components in the myelin sheaths (Ishizuka, 1997
; Merrill et al., 1997
). Deletion of ceramide galactosyltransferase (CGT), which is required for the synthesis of GalCer and sulfatide, leads to dysmyelinosis and death of CGT knockout mice (Bosio et al., 1996
; Coetzee et al., 1996
). It has been found that a marked decrease of sulfatide levels in the brain is associated with AD pathology (Han et al., 2002
), and that sulfatides are specifically associated with apoE-containing HDL-like particles (Han et al., 2003
). In the adult brain, apoE/lipoprotein particles are synthesized and secreted primarily by astrocytes (Pfrieger, 2003
; Puglielli et al., 2003
). ApoE particles then acquire sulfatides from the myelin sheaths likely through a “kiss and run” mechanism (Han, 2007
). These sulfatide-containing apoE particles can be internalized by neurons via lipoprotein receptors and then hydrolyzed in neuronal lysosomes, allowing intracellular release of free cholesterol, sulfatide and other lipids (Pfrieger, 2003
; Han, 2007
). A function for LRP1 in brain lipid metabolism has been postulated (Han, 2007
), but direct biological evidence had been lacking. Our findings provide direct evidence that deletion of LRP1 significantly alters the brain levels of sulfatide, GalCer, and cholesterol, all of which are critical for the integrity of neuronal membranes, dendritic spines and synapses. Interestingly, triglyceride was also decreased in the LRP1 knockout mice. Previous studies have demonstrated that apoE is associated with triglyceride metabolism in the nervous system (Cheng et al., 2007
). It has been reported that triglycerides account for less than 1% of the total lipids in mouse brain (Su et al., 1978). However, the exact function of triglyceride in the brain is not clear. It is generally accepted that brain energy metabolism was solely dependent on the glucose. However, increasing evidence shows that brain also used other energy resources, such as ketone, under the conditions of inadequate glucose availability or increased energy demands (Prins, 2008). Although brain cannot directly utilize fatty acids as energy source, they can be converted to ketone and utilized by the brain. The glycerol component of triglycerides can be converted into glucose, via gluconeogenesis, and used as an energy source (Cherrington et al., 1991
). Although triglyceride is not abundant in the brain, it is tempting to speculate that triglyceride may be utilized as an alternative energy source under certain conditions.
Dendritic spines are tiny protrusions along dendrites, which are essential for excitatory synaptic transmission. A loss or alteration of spines has been described in AD patients and in AD mouse models (Knobloch and Mansuy, 2008
). We also found significant decrease in dendritic spine density in the hippocampal and cortical regions of LRP1-KO mice at 18 months of age. Synaptic degeneration appears to be an early event in the pathogenesis of AD (Mashliah et al., 2001
; Scheff et al., 2007
) and synaptic impairment is likely to be the major contributor to memory loss in AD (Shankar and Walsh, 2009
). These dendritic spine degeneration and synaptic loss in LRP1 knockout mice are likely due to the impairment of lipid metabolism. We found that while lipid metabolism is impaired at 12 months of age in LRP1 forebrain knockout mice, degeneration of dendritic spines and synapses occurs only at older ages (18 months). Changes in the metabolism of other LRP1 ligands may also contribute to these defects. For example, thrombospondins, secreted by astrocytes, promote CNS synaptogenesis in vitro and in vivo (Christopherson et al., 2005
). Further, tissue plasminogen activator (tPA) plays an important role in the LTP and synaptic growth in the hippocampal mossy fiber pathway (Baranes et al., 1998
). Since both thrombospondins and tPA are ligands of LRP1, potential changes in their metabolism may also contribute to synapse loss and behaviral deficits in LRP1 knockout mice.
The most important feature of AD pathology is neurodegeneration. In most amyloid model mice, neuronal loss was reported only after the dramatic occurrence of amyloid plaques (Duyckaerts et al., 2008; Bredesen et al., 2009
). In our LRP1 knockout mouse model, we observed significant neurodegeneration in the hippocampus and cortex in the absence of amyloid pathology only at 24 months of age but not at 18 months of age. These pathological events parallel the progressive synaptic loss and neurodegeneration found in human AD brains (Shankar and Walsh, 2009
). The neuroinflammation observed in LRP1 forebrain knockout mice further indicates that neuropathology in the aged brain can occur in parallel with spine/synaptic loss in the absence of amyloid pathology.
The NMDA and AMPA receptors play essential roles in long-term potentiation (LTP), which is widely considered a major cellular mechanism underlying learning and memory. LTP is impaired in plaque-bearing AD transgenic mice and in the brain slices that were treated with Aβ peptides (Querfurth and LaFerla, 2010
). Disruptions of ion currents in the postsynaptic glutamate receptor result in part from the loss of surface NMDA receptors (Snyder et al., 2005
) or AMPA receptors (Hsieh et al., 2006
). Interestingly, we found that LRP1 deletion selectively decreases the protein levels of NMDAR1 and GluR1 in the brain and in primary cultured neurons. The decreased levels of NMDAR1, but not GluR1, can be rescued by addition of cholesterol to cultured neurons, suggesting that both cholesterol-dependent and independent mechanisms could contribute to the reduced levels of these glutamate receptor subunits in LRP1-deficient neurons. Interestingly, a previous study has demonstrated that addition of cholesterol to cultured neurons strongly enhances the number and efficacy of synapses (Mauch et al., 2001
). Conversely, depletion of cholesterol/sphingolipid leads to instability of surface AMPA receptors and gradual loss of synapses and dendritic spines (Hering et al., 2003
). The exact relationship between the loss of dendritic spines/synapses and selective glutamate receptors in LRP1 forebrain knockout mice requires further investigation.
Our current study defines a critical role for LRP1 at synapses in the adult brain. Interestingly, a previous report by Herz and colleagues demonstrated an important role for LRP1 in synaptic transmission by regulating the function of NMDAR and PSD-95 at the postsynapses (May et al., 2004
). In that study, LRP1 deletion in the SynI-Cre model was detectable much earlier and throughout the entire CNS. In contrast, with our current CamKII-Cre model, LRP1 deletion started between 3 to 6 months of age and gradually peaked at around 12 months of age and was restricted to the forebrain regions. Unlike the Syn-Cre/LRP1 mutant mice, which mostly died prematurely between 6 and 9 months of age, the CamKII-Cre/LRP1 mutant mice live to old age, which allows us to analyze the functon of LRP1 in the adult brain in an age-dependent manner. SynI-Cre/LRP1 mutant mice develop severe behavioral and motor abnormalities including hyperactivity, dystonia and systemic tremors starting around 3 weeks after birth. This more severe and earlier phenotype emphasized a crucial role for LRP1 in motor circuitry and muscle control, but effectively precluded examining another critical role for LRP1 in the adult CNS, i.e. maintaining brain lipid homeostasis and associated age-dependent synaptic and neuronal integrity as we have described here.
In summary, this study uncovers a biological function for LRP1 in modulating brain lipid metabolism and the integrity of dendritic spines, synapses and neuronal viability. These age-dependent events strongly resemble the neuropathology seen in human AD brains. Our results also provide important insights into how apoE and apoE receptors participate in brain lipid metabolism, which is critical for maintaining the integrity and function of brain synapses. Since the ε4 allele is the only genetic risk factor for late-onset AD, future studies should explore how the apoE alleles (E2, E3, E4) differentially affect LRP1 function with age.