These experiments revealed that ATP acts as a mitogenic signal for neural stem and progenitor cells, and may do so in an autocrine fashion. We found that when maintained as mitotically-active neurospheres, neural progenitor cells exhibited both ATP release and purinergic receptor-activated calcium mobilization. Real-time bioluminescence imaging of ATP release revealed that the progenitor cells themselves were the source of ATP, which they released in brief burst events. External addition of ATP or its analogs increased the mitotic index and rate of neural progenitor cells, whereas P2Y antagonists suppressed both neurosphere expansion and the mitotic index of cells within those spheres. Both ATP release and purine-activated calcium responses were retained over several months of repetitive passage, as was the reversible suppression of neurosphere expansion by P2Y antagonists. Strikingly, both the mitotic competence and multilineage potential of the cultured neural progenitor cells were restored upon P2Y antagonist removal. Indeed, the relative fractions of neurons, astrocytes, and oligodendrocytes that developed after plating of neurospheres were essentially unchanged in controls versus antagonist treated cultures. This contrasted to the expansion associated with other described positive regulators of neural progenitor cell expansion, such as sonic hedgehog, which have typically been associated with a sustained loss of neuronal differentiation competence (Wechsler-Reya and Scott, 1999
Kriegstein and colleagues (Weissman et al., 2003) demonstrated that calcium waves and attendant ATP release, with consequent P2Y1 receptor activation, accompanied radial cell-derived neurogenesis in cultured slices of the developing rat forebrain. This model permitted an assessment of BrdU incorporation for only 1 hour after ATP exposure, which was sufficient only to demonstrate accelerated DNA replication in the presence of ATP. In our present report, we noted that proliferation was indeed sustained for at least 5 days in the presence of ATP, indicating the competence of purinergic signaling in mediating progenitor cell proliferation and expansion. In addition, whereas Weissman et al identified primarily P2Y1-mediated purinergic transmission among radial cells, whose responses were thereby limited to ATP, we found that NSCs expressed P2Y-1, 2 and 4 receptors, and were hence sensitive to both ATP and UTP. These observations suggest a greater heterogeneity of purinergic signals, and hence of purine-mediated functions, in neural progenitor cells than revealed by slice culture. In this regard, real-time bioluminescence imaging of of ATP release by neurospheres revealed that neural progenitor cells were themselves the sources of local ATP. In particular, we noted that ATP was released by individual cells within each neurosphere in brief, evanescent bursts, that were exhibited by only a minor fraction of the cells, and through which ATP was effectively made available as a local and autocrine mitogen. This pattern of release was of especial interest since P2Y receptors can be desensitized for as long as 2 hrs after activation. Because purinergic receptors exhibit rapid desensitization to sustained agonist exposure, such brief bursts of transmitter release might then better stimulate proliferation than a more sustained stimulus, to which the cells might quickly become refractory (Burnstock, 2002a
). As a result, single brief bursts of ATP followed by local receptor inactivation suggest a burst-suppression model by which local populations of neural progenitor cells might be effectively synchronized and brought under the mitotic direction of single cells pacing the time course of ATP release.
Several lines of work have provided evidence for the importance of transient changes in Ca2+
. For example, Spitzer and co-workers showed that naturally occurring patterns of Ca2+
transients encoded neuronal differentiation (Spitzer et al., 2004
). Following closure of the neural tube, spinal cord neurons exhibit repeated Ca2+
transients produced by Ca2+
dependent action potentials. Pharmacological manipulations showed that neuronal differentiation was altered when Ca2+
transients were eliminated by blocking Ca2+
influx. Conversely, re-imposing different frequency patterns of Ca2+
elevation were sufficient to promote neuronal differentiation, including normal neurotransmitter expression and channel maturation (Gu and Spitzer, 1995
We have previously identified point-source bursts of ATP in cultured astrocytes, which exhibit a channel-mediated efflux of cytosolic ATP (Arcuino et al., 2002
). This suggested a role for purinergic signaling in the functional maintenance of the astrocytic syncytium. In this regard, astrocytes have been implicated in the support of neurogenesis from progenitor cells in neurogenic regions of the adult brain (Lim et al., 2000
) (Gage, 2000
) (Song et al., 2002). As a result, the paracrine activation of neural progenitor cells by their astrocytic neighbors might then serve to maintain their undifferentiated self-renewal. As such, astrocytic ATP may be viewed as negatively regulating neurogenesis, by maintaining the mitotic activity of resident precursors, while suppressing their terminal neuronal or glial differentiation.
In accord with this model, both serum addition and substrate anchorage substantially reduced the ability of neural progenitors to release ATP (), consistent with the differentiative effects of both serum and substrate on neural progenitor cells (Goldman et al., 1992
) ((Reynolds and Weiss, 1992
). Indeed, nucleotide-mediated signaling was rapidly lost during neuronal differentiation: Purinergic receptor expression was uniformly down-regulated early in the process of neuronal differentiation, and exposure to receptor agonists failed to mobilize cytosolic calcium in MAP-2+
neurons. Together, these observations suggest that neural progenitor cells both release ATP, and respond to it with an increase in proliferation. As such, ATP appears to act through both autocrine and paracrine routes to regulate the division and proliferation of neural progenitor cells. The high ectonucleotidase activity of cultured neurospheres may serve to modulate this process by regulating ATP and ADP bioavailability, thereby allowing neurogenesis to proceed despite the active purinergic signaling needed for expanding the progenitor cell pool antecedent to neuronal differentiation. Indeed, to the extent that NTDPase activity may act to remove local ATP and ADP and hence diminish local purinergic signaling, the expression of NTDPase by neural progenitor cells may be critical to their production of neurons. In this regard, neurospheres typically generate a fraction of neurons peripherally, pare passu with the concurrent central expansion of uncommitted neural progenitor cells. It would be fair to note, however, that our observation does not provide direct evidence for the importance of ATP burst release vs tonic release of ATP. Technical limitations prevented us from discriminating between the two pathways of ATP release. Nevertheless, our observations strongly suggest that the regulation of purinergic signaling may be critical to neural progenitor cell expansion.
ATP release and purinergic signaling may be required not only for developmental progenitor cell expansion and neurogenesis, but also to the persistent progenitor cells of the adult brain. The co-localization of both P2Y receptors and ectonucleotidase activity to regions of active mitotic progenitor cell expansion and neurogenesis in the adult brain (Braun et al., 2003
; Shukla et al., 2005
) is especially significant in this regard, given the apparent necessity of purinergic signaling to neural progenitor cell expansion in vitro. Just as neural progenitor cells appear to undergo ATP release as a means of both autocrine and paracrine purinergic activation of self-renewing divisions, their local production of NTDPase may serve as a brake on that process, clearing ATP and preventing uncontrolled expansion while establishing a permissive microenvironment for neuronal differentiation. By delimiting progenitor cell proliferation, the NTDPase-mediated clearance of ATP may thereby provide an important permissive condition for neurogenesis, and hence for the maintenance of local neurogenic niches. Indeed, the association of NTDPase activity with the capillary microvasculature (Braun et al., 2000a
) suggests that purinergic signaling may contribute to the angiogenic support of neurogenesis in adult neurogenic niches (Cleaver and Melton, 2003
; Louissaint et al., 2002
; Palmer et al., 2000
). As a result, progenitor cell-derived neurogenesis may require both active purinergic signaling and the negative regulation thereof.
Our data suggest that ATP burst-activated purinergic signaling may constitute a critical regulatory checkpoint for modulating the expansion and lineage commitment by neural progenitor cells. As such, its abrogation, through a suppression of either purine release or reception, may comprise a means of inhibiting undesired progenitor or progenitor cell expansion, as may be the case in neoplasias of the CNS (Burnstock, 2002a
). In contrast, its stimulation may provide a means of expanding progenitor cell populations, so as to provide an expanded cellular substrate for strategies designed to induce neurogenesis from endogenous progenitor cell pools. On this basis, the pharmacological regulation of purinergic signaling may permit us as a means of modulating neurogenesis in both the fetal and adult brain.