Cholesterol and apolipoproteins in Alzheimer's disease have become a rapidly developing-and sometimes confusing-area of investigation, but most of it suggests that lowering cholesterol might slow disease progression. A paper in tomorrow's Science presents a new, beneficial function for cholesterol in the brain that may help explain some previous data. Scientists led by Frank Pfrieger the Max-Planck/CNRS group in Strasbourg, France, report that neurons need cholesterol secreted by glial cells to form and maintain functional synapses.

"Our work introduces a new connection between synapse formation and cholesterol metabolism that was not realized before," Pfrieger said. "The main hypothesis from our findings is that CNS neurons are able to synthesize a certain amount of cholesterol that supports their survival and possibly their growth, but at the same time they also require additional cholesterol for synaptogenesis, which they cannot synthesize themselves. This needs to be imported. We think that the centers of synthesis are glial cells. It is known that astrocytes make ApoE and secrete lipoproteins, but until now nobody knew why," he continues.

This work began in 1997, when Pfrieger, then in Ben Barres' laboratory at Stanford University in California developed a culture system for purified retinal ganglion neurons. He observed that neurons formed functioning synapses only once astrocytes were added to the culture. Removing the astrocytes caused the neurons to lose those synapses again. Follow-up papers suggested that astrocytes control synapse formation by secreting a mysterious soluble factor (Ullian et al., Nagler et al.).

The present paper identifies this factor as cholesterol. The researchers treated cultured neurons with elution fractions from fractionated glia-conditioned medium, recorded spontaneous synaptic activity in these neurons, and further purified those fractions that induced increased activity. The researchers also used two-D gel electrophoresis and protein sequencing by nanospray mass spectrometry to identify a complex containing ApoE in the membranes of neurons treated with glial-conditioned medium. Their initial hunch that ApoE might be the elusive factor proved wrong but their next guess-cholesterol-proved right.

Further experiments showed that cholesterol increased the frequency of spontaneous postsynaptic currents and that reducing cholesterol with the drug mevastatin eliminated this effect, as did inhibiting lipoprotein uptake with RAP, a chaperone known to inhibit ligand (i.e., ApoE-cholesterol) binding by LDL receptors. Finally, the scientists show cholesterol increases by eightfold the number of autapses (those are synapses an individually cultured neurons makes with itself) and by 10-fold the quantal content of neurotransmitter release per neuron.

"The simplest explanation for these findings is that cholesterol bound to ApoE-containing lipoprotein particle is released by astrocytes and taken up by neurons, where it then promotes an increase in synapse number," write Barres and Stephen Smith in an accompanying Perspectives article.

Neurons probably require glial cholesterol as building material for the assembly of presynaptic components such as synaptic vesicles and release sites, but an additional role in signal transduction is also possible, Pfrieger said. These findings fit in with the successive waves of cell-type differentiation in the embryonic brain and might explain why synaptogenesis begins only after astrocytes have developed.

The relevance of this work to Alzheimer's remains to be investigated. Given that ApoE4 is associated with increased risk for late-onset AD, one experiment would be to test whether the different isoforms of ApoE affect synapse formation differently in this culture system. It is unknown whether adult neurons depend on glial-derived cholesterol for synapse maintenance or for the formation of new ones during learning and memory formation. Intriguingly, the LDL-receptor related protein has been implicated in synaptic plasticity in hippocampal slices (Zhuo et al.). In trying to understand this question, it is important to keep in mind that most brain cholesterol must be synthesized within the brain rather than being imported from the blood, says Pfrieger (Danik et al.).—Gabrielle Strobel

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  1. Mauch et al., in a well-designed, interesting study, demonstrate the
    importance of cholesterol to the formation of electrically functional
    synapses in cultured retinal ganglion cells. They demonstrate that retinal ganglion cells attain their normal morphology when
    cultured under serum-free defined conditons in the absence of glial cells. Their synaptic number and activity, however, is low under these conditions. The addition of glial-conditioned media increases synaptic activity and number in these cells. It is shown that fractions of this media containing ApoE-containing lipoproteins account for the effects of the glial-conditioned media in increasing synaptic activity and number. These effects can be mimicked by adding exogenous cholesterol (without ApoE) and inhibited by inhibiting cholesterol synthesis by glia.

    The findings demonstrate that adequate levels of cholesterol are required by developing retinal ganglion neurons in culture for attaining normal numbers of functioning synapses.

    The findings imply that normal levels of cholesterol in the brain are critically important during development for normal synapse formation and function. How these findings relate to the importance and role of specific molecules in the brain (in vivo) in this process or to disease conditions is not yet clear. A future study that might be useful would be to test whether the same effects observed in the Mauch et al. study can be seen in mouse retinal ganglion cells cultured on glia derived from apoE -/- vs. wild-type mice.

    Whether levels of cholesterol or its delivery to neurons ever becomes so
    limiting in the brain (as in the culture conditions studied) to result in cholesterol depletion enough to cause decreased synaptic number or efficacy is unknown. ApoE is the most abundant apolipoprotein in brain and it can deliver cholesterol to neurons and glia. Most but not all evidence suggests that synapse formation and number is normal in the CNS of ApoE -/- mice after birth and into development. There may be other molecules and redundant pathways that allow neurons in the CNS to acquire enough cholesterol in the absence of ApoE in vivo to attain normal synaptic number and function. No abnormality in intellect or brain development has been reported in human individuals who lack ApoE. It is certainly conceivable that cholesterol availability becomes abnormal under certain conditions in the aging brain and that ApoE, its isoforms, or other molecules important to cholesterol synthesis and delivery could specifically influence synaptic function. Further studies will be needed to sort out these issues.

  2. Please see the following comment ont this article and its accompanying commentary: Cholesterol's role in synapse formation Koudinov and Koudinova Science 2002.

    View all comments by Alexei Koudinov
  3. The compelling data by Mauch et al. affirms the critical nature of cholesterol in the CNS, particularly the developing CNS. As with most very good research, the experiments lead to important answers and provide the foundation for more questions.

    These authors clearly demonstrate that cholesterol/apolipoprotein E are essential for synaptogenesis and probably for appropriate production and transportation of organelles (i.e. vesicles) necessary for neurotransmission. Coupled with the understanding that a large portion of a developing growth cone is cholesterol, this suggests that a pathologic condition or centrally acting agent, which disrupts cholesterol metabolism, could be devastating to a human infant, both pre- and postpartum.

    If one accepts the concept that there is plasticity to synaptic contact in the mature CNS in that synaptic contacts may be formed, dissolved, and reformed as necessary, then directly altering cholesterol metabolism in the adult brain could also have deleterious consequences.

    Some believe that sprouting and an attempt to re-connect lost circuits occurs in neurodegenerative disorders. If that is so, the effect of altering cholesterol metabolism could depend on whether or not re-connection is faithful. It is possible that misconnection of synaptic circuitry (innervating the wrong target) could be worse than the result of the neurodegeneration alone. In this case it may be appropriate to attempt to attenuate such misguided synaptogenesis. These are only possibilities that await further clarification.

References

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Further Reading

Papers

  1. . Control of synapse number by glia. Science. 2001 Jan 26;291(5504):657-61. PubMed.
  2. . Glia-derived signals induce synapse formation in neurones of the rat central nervous system. J Physiol. 2001 Jun 15;533(Pt 3):665-79. PubMed.
  3. . Role of tissue plasminogen activator receptor LRP in hippocampal long-term potentiation. J Neurosci. 2000 Jan 15;20(2):542-9. PubMed.
  4. . Brain lipoprotein metabolism and its relation to neurodegenerative disease. Crit Rev Neurobiol. 1999;13(4):357-407. PubMed.

Primary Papers

  1. . CNS synaptogenesis promoted by glia-derived cholesterol. Science. 2001 Nov 9;294(5545):1354-7. PubMed.
  2. . Neurobiology. Cholesterol--making or breaking the synapse. Science. 2001 Nov 9;294(5545):1296-7. PubMed.