Many diseases that affect brain function involve changes of synaptic function, which is not surprising considering the central role of synapses in neuronal communication. The discovery of synaptic dysfunctions that correlate with brain diseases has motivated the use of the term synaptopathy, in order to imply that in these diseases synaptic function is affected (Brose et al., 2010). Considering the complexity of the brain, neurons and synapses it is not always possible to determine whether changes at the level of the synapse are the cause of a given disease or if they result from alterations in more basic neuronal functions.
Nevertheless, the list of brain disorders for which synaptic dysfunctions have been described is long and includes Autism, Parkinson’s disease, Schizophrenia, Alzheimer’s disease and Huntington’s disease.
Alzheimer’s disease (AD) is the most common form of dementia in the aged population, with 4.6 million new cases each year worldwide (Smith, 2006). AD is a neurodegenerative disease that is characterized by the loss of neurons, synapses as well as the presence of ‘senile plaques’ containing amyloid-β and ‘neurofibrillary tangles’ containing hyperphosphorylated tau protein (for reviews see (Arendt, 2009; Lee et al., 2001b;
Parameshwaran et al., 2008)). Several synaptic proteins have altered expression levels in AD. Synaptic dysfunction precedes neuronal loss in the development of AD and seems to affect especially excitatory synapses in cortex and hippocampus (Arendt, 2009). This is underlined by the fact that loss of VGLUT1 and VGLUT2 protein expression levels are correlated with the cognitive decline during AD (Kashani et al., 2007).
Huntington’s disease (HD) is a progressive late-onset neurological disorder caused by CAG-repeat expansions in the gene encoding huntingtin (HTT). It is characterized by abnormalities in motor coordination, cognitive impairments and psychiatric manifestations (Group, 1993). The clinical symptoms of perturbed motor coordination coincide with a loss of striatal medium-sized spiny neurons (MSNs) (for review (Cowan and Raymond, 2006)).
These inhibitory neurons receive excitatory input from the thalamus and from the cortex and it has been shown that an excess in NMDA-mediated excitation contributes to the loss of MSNs in HD. In HD, MSNs show an increase in surface expression of the NR2B subunit (Fan et al., 2007). Furthermore, the negative effect on the survival of MSNs is mediated by extrasynaptic NR2B containing NMDA receptors, whose downstream signalling cascades affect the neurotoxicity of the mutant HTT in MSNs (Milnerwood et al., 2010; Okamoto et al.,
2009). In contrast, activation of synaptic NMDA-receptor signaling has a neuroprotective effect in MSNs by promoting the formation of HTT inclusions (Okamoto et al., 2009).
Autism spectrum disorders (ASDs) represent a group of neurodevelopmental disorders characterized by atypical social behavior, disrupted verbal communication and unusual patterns of restricted interests and repetitive behaviors with varying severity (Association, 1994). Several cases of hereditary nonsyndromic cases of ASDs have been reported and include individuals with loss-o- function mutations in genes encoding Neuroligin3 and Neuroligin4 (Jamain et al., 2003; Südhof, 2008). Members of the Neuroligin family of cell adhesion molecules are important for proper synapse maturation and function (Varoqueaux et al., 2006). Interestingly, mouse models deficient for Neuroligin3 or Neuroligin4 mimic certain behavioral aspects associated with autistic phenotypes (Jamain et al., 2008; Radyushkin et al., 2009; Tabuchi et al., 2007).
While being far from comprehensive, this short overview of a few prominent cases illustrates that synaptic dysfunction is a hallmark of many brain disorders and that changes in the function, expression levels or subcellular localization of synaptic proteins can be diagnostic or causative for several aspects of these diseases. For this reason the characterization of the protein composition of synapses will be of great importance for a more complete understanding of their role in the physiology and pathology of the brain.
1.5 Synaptosomes
Brain tissue homogenization in non-ionic, iso-osmotic media allows the release of synaptosomes, which are resealed nerve terminals separated from the axon and dendrites (for review see (Whittaker, 1993)). The term synaptosome was coined by Victor Whittaker to describe an isolated, functional synaptic particle that contains mitochondria, possesses a transmembrane potential and retains many of the features of pre- and postsynaptic elements, such as SVs, an active zone and a postsynaptic density.
Using a combination of differential centrifugation and sucrose-density gradient centrifugation, synaptosomes were first enriched by Catherine Hebb and Victor Whittaker in 1957 (Hebb and Whittaker, 1958)(see Figure 6 for illustration). Since then, many variations of this subcellular fractionation protocol have been published. Nevertheless, the original sucrose-density gradient procedure yields synaptosomes of the highest purity (Whittaker, 1993). To date, it seems that almost all synapse subtypes, from any brain region, can yield synaptosomes (Whittaker, 1993). Notable exceptions are the large mossy fiber synapses, which might be disrupted under conditions used for preparation of other synaptosomes (Israël and Whittaker, 1965).
Since their discovery about 50 years ago, a great amount of knowledge about synapse structures and functions has been gained from studies on synaptosomes. For example, some of the first indications that Serotonin and substance P might serve as neurotransmitters in the CNS came from their abundance in synaptosome preparations (CLEUGH et al., 1964; Whittaker, 1959). Many aspects of the plasma membrane uptake of neurotransmitters and their precursors have been studied in synaptosome-derived plasma membranes (Kanner and Schuldiner, 1987). In addition, experiments on synaptosome-derived SVs were essential in providing direct evidence that the amino acid neurotransmitters glutamate, GABA and glycine are actively transported and stored in SVs (reviewed in (Maycox et al., 1990)). On September 1, 2010 a pubmed query using the term
“Synaptosome” returned 12698 entries. 928 articles contained “Synaptosome” in the title or abstract of which 290 were published between the years 2000 and 2010. This underlines that the synaptosome preparation was not only instrumental for studies of the molecular function of synapses in the past, but that it remains an important technique to study many aspects of synaptic function.
Figure 6: Overview of the preparation and contents of synaptosomes and synaptosome-derived fractions
This diagram illustrates a subcellular fractionation procedure for the preparation of crude synaptosomes (P2), gradient purified synaptosomes (B), synaptic plasma membranes (LP1B) and crude SVs (LP2). Intact synaptosomes, synaptic plasma membranes and isolated SVs are represented schematically.