Determining the intracellular localization of a protein is essential to understanding function, and therefore to the development of rational therapies for genetic diseases in which specific proteins are lost as a result of mutations. As described in the Introduction, a previous study [9
] using postmortem human brain tissue concluded that the ATM protein is located in the cytoplasm of human Purkinje neurons. Cytoplasmic localization implied that the function of ATM in Purkinje neurons is something separate from the established role of the ATM protein, which is the detection and repair of DNA damage. However, in the earlier work [9
], control studies using brain tissue from AT patients as a negative control were not employed, thus raising questions about the specificity of the ATM staining. Here, using an antibody previously demonstrated to specifically stain ATM in human neuronal-like cells in culture [12
], we show that ATM is predominantly localized in the nucleus of Purkinje neurons in postmortem tissue from the juvenile human brain. Importantly, our conclusion is supported by control experiments demonstrating the absence of staining in Purkinje neurons from AT patients, which represents the most conclusive evidence for the specificity of the staining. As such, this localization is consistent with the hypothesis that a major function of ATM in fully differentiated human Purkinje neurons is to detect nuclear DNA damage, as it is in all other cell types examined.
McKinnon and colleagues [26
] have shown that in mice, the ATM protein is required for triggering apoptosis in response to neuronal DNA damage during the period shortly after the stage of terminal differentiation. Importantly, however, this observation does not exclude an ongoing role of the ATM protein in Purkinje neurons at later stages (see [27
]). In humans, Purkinje neuron differentiation extends from the late fetal period into the first year of life [28
]. Therefore, in addition to a role in triggering apoptosis in response to DNA damage in early brain development, our observation of nuclear ATM localization in human Purkinje neurons in normal individuals 14 and 17 years of age is consistent with the hypothesis that ATM also plays an ongoing role in sensing DNA damage in Purkinje neurons long after the terminal differentiation stage.
It is important to note that while ATM staining intensity was clearly highest in the cell nucleus, we also observed some ATM staining in the cytoplasm of Purkinje neurons as well. This staining may correspond to ATM in cytoplasmic vesicles, as described by others [29
]. While the intensity of cytoplasmic staining was very light compared to the nuclear staining, given the large volume of cytoplasm in human Purkinje neurons, this light staining could represent a substantial amount of the total ATM protein. However, the functional significance of this cytoplasmic ATM, if any, remains to be determined.
While the primary goal of this work was to address the localization of ATM in Purkinje neurons, we also examined ATM staining in other cerebellar neurons. Notably, very weak staining was observed in granule cells, but strong staining was observed in interneurons within the granule cell layer, as well as in neurons in the molecular layer. It is becoming increasingly clear that the cerebellar cortex contains a variety of morphologically and biochemically distinct types of interneurons [16
], and additional studies would be necessary to fully categorize the ATM staining in each specific type.
A full description of ATM staining in the rest of human brain is beyond the scope of this work. However, consistent with the widespread expression of ATM mRNA in the human brain [32
], we detected ATM protein expression by Western blotting in multiple brain regions (cerebellum, pons, cerebral cortex, hippocampus, and thalamus) and detected predominantly nuclear ATM staining in neurons in the human cerebral cortex (data not shown). Since the neuropathology observed in AT patients primarily affects the cerebellum (although cells in the spinal cord, dorsal root ganglia, and basal ganglia are also affected [3
]), our observations provide further evidence that ATM expression is not limited to brain regions or cell types that are affected in AT patients, and emphasize the importance of understanding why Purkinje neurons are so severely affected in AT patients.
The MRN-Complex and Topoisomerase I are also Concentrated in Human Purkinje neurons
In contrast to ATM, we did not have access to human tissues from patients lacking the MRN proteins or TOP1 as negative controls for antibody specificity. With regard to TOP1, such material will never be available, since TOP1 is an essential protein. In lieu of negative control tissues, we relied on the concordant results of multiple independent antibodies to demonstrate staining specificity. This approach is analogous to the use of multiple siRNAs directed against different regions of an mRNA to demonstrate specificity [33
]. Also, our localization of the MRN proteins in human Purkinje neurons is completely consistent with the observations of Jacobsen et al. [19
]. With regard to TOP1, in addition to obtaining identical results using two independently generated monoclonal antibodies, our observation that TOP1 is localized to the nucleolus and nucleoplasm is consistent with other studies on the distribution of this enzyme within the nucleolus [23
], and nucleus, including observations of GFP-tagged TOP1 in living human cells [24
]. It should be noted that in one study [34
], cytoplasmic TOP1 staining was observed in the mouse Purkinje neurons. However, in another study [35
] TOP1 staining was observed in the nucleolus of postnatal rat Purkinje neurons, consistent with our findings. Whether the discrepant observations [34
] represent a species difference, a methodological difference, or non-specific staining remains to be determined.
The fact that the MRN complex and ATM act together in a double-strand break repair pathway in human cells is well established, although the exact details of how this pathway functions is the subject of ongoing investigations [2
]. Our observations of high levels of ATM and MRN proteins in Purkinje neurons are consistent with the hypothesis that this same pathway functions to repair endogenous double-strand breaks (perhaps arising from closely apposed single-strand breaks) in human Purkinje neurons. However, our observation that ATM is present in the nucleolus of Purkinje neurons, while the MRN proteins are essentially excluded from this compartment, raises the possibility that ATM may have additional functions in the nucleolus of Purkinje neurons which are independent of the MRN complex, although further experiments will be necessary to test this possibility.
In terminally differentiated cells such as neurons, the main role of TOP1 is presumably in transcription, and in particular transcription of ribosomal DNA [23
]. The strikingly high level of TOP1 we observed in Purkinje neurons compared to other cerebellar neurons indicates that these cells have an elevated requirement for this enzyme, perhaps due to a high demand for ribosomal RNA synthesis. TOP1 can become covalently attached to the DNA, forming TOP1 cleavage complexes (TOP1ccs) [36
], and many types of endogenous DNA modifications can cause TOP1ccs [37
]. The observation that spinocerebellar ataxia with axonal neuropathy (SCAN1) results from mutations in the TDP1
], which encodes a protein involved in the repair of TOP1ccs [38
] indicates the importance of repair of such complexes in preventing ataxia. In view of the high levels of TOP1 in Purkinje neurons, it follows that these cells would be at elevated risk for TOP1ccs, and thus the loss of ability to repair such complexes would be particularly severe in these cells. If ATM is involved in the repair of TOP1ccs, this would provide a possible explanation of why Purkinje neurons are specifically affected in AT patients. Additional functional studies are necessary to address this point.