Neurons and neuronal cell lines appear to be unique in that a substantial fraction of their ATM protein is cytoplasmic, a conclusion supported by three different lines of evidence: subcellular fractionation, immunocytochemistry and the distribution of an exogenous GFP-ATM fusion protein. The prominence of this cytoplasmic component is cell-type specific as neither spleen, nor thymus nor cell lines of non-neuronal origin have significant amounts of ATM outside of their nucleus.
We postulate that this non-nuclear ATM is different in its function as well as in its location. The biochemical and physical relationships among ATM, ATR, VAMP2 and synapsin-I, coupled with the defects in LTP and spontaneous dye release observed in ATM-deficient neuron are strong evidence that cytoplasmic ATM plays cellular roles in neurons that are unrelated to the DNA damage response. Cytoplasmic ATM shows no evidence of activation as assessed by phosphorylation at S1981. Yet, others have speculated that ATM kinase activity unrelated to the phosphorylation of S1981 exists23, 24
, and the ATM-dependent phosphorylation of synapsin-I at S656 would tend to support this suggestion.
Both VAMP2 and synapsin-I are best known for their roles in the pre-synaptic nerve terminal, and we have shown that ATM protein is found in synaptosomal preparations. It is unlikely, however, that the function of cytoplasmic ATM is restricted to pre-synaptic terminal as immunocytochemistry also reveals robust staining in the cell soma and dendrites. The actions of cytoplasmic ATM and ATR appear to be symmetrical. We show that all four proteins can physically associate with one another in the cytoplasm, but while the ATM kinase targets synapsin-I, ATR targets VAMP2. The involvement of the ATR kinase in the cytoplasmic function of ATM is intriguing as a partial deficiency of ATR leads to devastating neurological consequences25
The subtle nature of the synaptic changes in ATM-deficient neurons suggests a regulatory role for cytoplasmic ATM in neuronal activity rather than a central function in synaptic connectivity. It seems likely that either phosphorylation of VAMP2 or its binding to ATM would sterically interfere with its normal function in vesicle docking and fusion. Perhaps ATM holds a pool of VAMP2 and/or synapsin-I in reserve to be released as needed. Alternatively, ATM may assist in the transport of VAMP2 and synapsin-I by keeping them in a protected stat, consistent the distribution of the non-phosphorylatable proteins in cultured neurons.
In the aggregate, these novel associations of ATM with synaptic function provide new and unexpected explanations for the severe neurological symptoms in children with A-T, and possibly open new avenues of intervention specifically related to cytoplasmic ATM function26
. It would appear that in conjunction with the cell cycle related cell death27
A-T neurons are subject to a synaptic dystrophy that can only exacerbate the damage caused by cell loss.