The purpose of undertaking this tissue culture study was not to replicate disease progression in the Ppt1 deficient brain, but rather to assess intrinsic defects in
Ppt1−/− glial biology. Indeed, in addition to detailing defects in the morphology and survival of neurons, this study is the first to characterize
Ppt1−/− glia in vitro
. Our data reveal that not only do
Ppt1−/− microglia and astrocytes exhibit a range of abnormal phenotypes, they also appear capable of harming neurons, especially if these neurons are Ppt1 deficient. Whilst
Ppt1−/− astrocytes predominantly affect neuronal morphology, the presence of
Ppt1−/− microglia alone was enough to impact neuron survival, a negative influence that was greater if both
Ppt1−/− astrocytes and
Ppt1−/− microglia were present, resulting in pronounced neuron loss. These data suggest that glial cells are more affected by Ppt1 deficiency than previously anticipated, and this may directly influence neuron survival in CLN1 disease and it will be important to explore this also occurs in vivo. However, as discussed below, these defects in Ppt1 deficient glia are quite distinct from those we recently reported in similar experiments in CLN3 disease [
35], another member of this group of disorders in which a negative influence of functionally compromised glia upon neuronal survival is evident. Taken together, these data provide further evidence that although these disorders broadly share similar features, they may differ markedly in how individual cell types are impacted by disease.
Effects of Ppt1 deficiency upon glial biology
Compared to the morphologically attenuated glial activation that is evident in CLN3 disease [
35], mouse models of CLN1 disease exhibit much more pronounced and early onset activation of both astrocytes and microglia in the same CNS regions that later display the most neuron loss. This includes the thalamocortical system [
23], cerebellum [
29], and long before these events occur in the brain this glial activation is also evident at all levels of the spinal cord [
43]. These reactive events are accompanied by a pronounced upregulation of chemokines and cytokines in vivo [
29], that can be normalized by a combination of brain directed gene therapy and anti-inflammatory drugs [
28]. Our data demonstrate that cultured
Ppt1−/− astrocytes are very responsive to pharmacological stimulation (Figs.
1), and
Ppt1−/− microglia appear morphologically more activated even under basal unstimulated conditions (Fig.
5). This is quite different to the properties of cultured
Cln3−/− astrocytes and microglia that both respond slowly and incompletely [
35], mirroring the relative extent of morphological transformation of these cell types with disease progression in murine CLN1 and CLN3 diseases in vivo [
23,
35,
37,
38]. Another marked phenotypic difference between glia isolated from mouse models of these two forms of NCLs is the reduced secretion of chemokines and cytokines by
Cln3−/− astrocytes and microglia in culture [
35], compared to the elevated secretion of a subset of these factors by
Ppt1−/− astrocytes (Fig.
2) and microglia (Additional file
1: Figure S1), data that mirrors in vivo findings for much broader elevation of such factors [
29]. Any comparison between in vitro and in vivo studies must be made with caution. However, there are differences between these expression profiles in terms of which chemokines are upregulated. These may reflect the markedly different ages of the mice when these studies were performed, the inherent differences in a tissue culture environment and the living brain, as well as the pharmacological stimulation used in the current in vitro study.
Another fundamental difference between data from Cln1 and Cln3 glia, is that compared to the disruption of intermediate filaments seen in
Cln3−/− astrocytes [
35], the current study found no evidence for any similar cytoskeletal defects in
Ppt1−/− astrocytes (data not shown). Taken together, these data emphasize that although glia from both of these mouse models of NCL display a variety of abnormal phenotypes in culture, and that these consistently affect astrocytes more than microglia, the nature and extent of these glial defects differ markedly between CLN1 and CLN3 disease.
One of the most striking findings of the current study is the compromised survival of
Ppt1−/− astrocytes in all culture conditions, a phenotype not exhibited by
Cln3−/− astrocytes in vitro [
35]. It will be important to determine if this vulnerability of
Ppt1−/− astrocytes is related to abnormal calcium signalling properties that they display (Fig.
4), but it might be expected that these raised cytoplasmic calcium levels would contribute to their death. It will be crucial to determine if
Ppt1−/− astrocyte survival is also impaired in vivo to a similar extent that we have observed in vitro. Although there is already evidence for fewer astrocytes expressing S100β and glutamine synthetase in
Ppt1−/− mice during the later stages of the disease [
29], which points towards a loss of these astrocyte populations in vivo, this issue is yet to be fully resolved. Our in vitro findings do provide further corroborative evidence for an inherent vulnerability of
Ppt1−/−astrocytes, and this would be expected to adversely impact neurons and their survival during CLN1 disease progression. However, the exact mechanisms underlying such cell-specific vulnerability of astrocytes remain unclear and will need to be investigated in subsequent in vivo studies.
Effects of Ppt1 deficiency upon neurons
CLN1 disease is perhaps the most profoundly neurodegenerative form of NCL, with near total cortical neuron loss at autopsy [
18]. However, the mechanisms by which a deficiency in a de-palmitoylating lysosomal enzyme leads to this remarkably dramatic neuron loss remain unclear. Previous studies culturing neurons derived from
Ppt1−/− mice surprisingly did not reveal any overt defect in their survival, or firing properties, but did find alterations in the synaptic vesicle pool size [
47]. These are suggestive of an early stage synaptic pathology, and
Ppt1−/− mice do go on to display progressive changes in synaptic organization within the thalamocortical system [
24]. Our data in this study provide new evidence that
Ppt1−/− cortical neurons show compromised survival in our culture conditions (Fig.
6c), display a markedly smaller cell soma (Fig.
6d) and compromised neurite organization in vitro, that involves secondary and tertiary neurites rather than those arising directly from the soma (Fig.
7d-f). This neuron loss was most pronounced for GABAergic interneurons (Fig.
6b), consistent with in vivo data from these
Ppt1−/− mice [
3,
23]. Taken together, these data reveal that Ppt1 deficiency does not by itself result in the same profound extent of neuron loss seen in vivo, suggesting that other non-neuron-intrinsic mechanisms may operate to influence neurodegeneration in this disorder.
Assessing the glial contribution to neuron loss
There is an increasing body of evidence that glial activation and/or dysfunction have an active role in the pathogenesis of several neurodegenerative disorders [
1,
36,
40,
44]. Although not necessarily themselves instigators of neurodegeneration, any disruption of the normal support functions served by astrocytes or microglia may plausibly lead to neuron dysfunction or loss. The concept that glial cells can contribute to disease severity and accelerate its progression is perhaps most advanced in ALS and Parkinson disease, but it is becoming apparent this principle also extends to several lysosomal storage disorders. Lysosomal dysfunction and disrupted autophagy were thought to lead to the toxic role of astrocytes in multiple sulfatase deficiency, with the suggestion that a genetic defect in astrocytes alone was sufficient to drive the disease [
11]. Microglial activation has also been suggested to contribute adversely to the pathogenesis in various LSDs including MPSI, MPSIIIB, Sandhoff disease and GM2 gangliosidosis [
22,
33,
48], but the extent to which this happens in the NCLs is less clear.
The close correlation between the sites where early localized glial activation occurs in every form of NCL, and the extent of subsequent neuron loss [
9,
34], has always begged the question of whether these events may be mechanistically related to one another. So far, the role of glia in these disorders has been investigated in most depth in CLN3 disease, and there is recent evidence for a negative influence of Cln3 deficient microglia [
51] and astrocytes [
4] upon neurons. We have recently extended these observations, using very similar primary culture and co-culture systems used in the present study [
35]. This approach revealed a variety of defects in the biology of
Cln3−/− microglia and astrocytes, and when grown in co-cultures, these dysfunctional glia harmed healthy neurons and resulted in the death of
Cln3−/− neurons [
35]. These data prompted us to investigate whether there was any influence of
Ppt1−/− astrocytes and microglia upon neuron health in CLN1 disease.
Despite the marked differences in the phenotypes displayed in vitro by
Ppt1−/− glia (this study) and
Cln3−/− glia [
35], a consistent feature is that astrocytes and microglia deficient in either gene exert a negative influence of upon neuronal morphology and survival. The relative contributions of either
Cln3−/− astrocytes or microglia to these processes are yet to be defined, as are the underlying mechanisms, but the current study has provided the first insights into the negative influence of glia in CLN1 disease. Our data reveals that this adverse impact varies between cell types, with
Ppt1−/− astrocytes only exerting a moderate influence on neuronal morphology (Fig.
8), whereas
Ppt1−/− microglia are capable of inducing the death of neurons (Fig.
9), especially if these neurons are themselves Ppt1 deficient. These differences likely reflect how each of these cell-types are compromised by Ppt1 deficiency, and it will be important to investigate these mechanisms by transcriptional profiling. Whatever the underlying mechanisms may be, this adverse influence appeared to be exacerbated when both
Ppt1−/− astrocytes and
Ppt1−/− microglia were present (Fig.
11). This is consistent with recent data suggesting that under certain conditions microglia may prime astrocytes to become neurotoxic [
25]. However, the specific factors implicated in this priming mechanism were not all upregulated by
Ppt1−/− microglia, and it may be that different mechanisms operate in CLN1 disease. However, data from our co-cultures of
Ppt1−/− astrocytes and
Ppt1−/− microglia together (without any neurons present) suggest that these mutant astrocytes appear to drive these
Ppt1−/− microglia to become further activated (Fig.
10). Indeed, it seems plausible that the toxic effect of
Ppt1−/− microglia (Fig.
9) may be exacerbated by the consequence of impaired
Ppt1−/− astrocyte function and/or survival to create conditions that promote neuron loss, perhaps via the increased release of IL-1β from microglia (Additional file
1: Figure S1), but this awaits experimental verification. Previous data had suggested astrocyte activation was beneficial in this disorder (Macauley et al., 2011), since disease progression was accelerated in
GFAP−/−/Vimentin−/−/Ppt1−/− mice in which glial activation is genetically suppressed. This is in marked contrast to data from our co-culture systems that suggest a negative influence of
Ppt1−/− astrocytes upon neuron health (Figs.
8,
11), and it will be essential to validate these effects in vivo.
In order to address such issues, we are already generating mice in which Ppt1 can be inactivated in a cell type-specific manner. Such approaches would normally be complicated by the release of Ppt1 enzyme from genetically unmodified cells to cross correct the cell type in which Ppt1 has been inactivated. However, the generation of mice expressing a biologically active membrane tethered Ppt1 enzyme that is not secreted [
42] provides important proof of principle for this strategy, which will be employed in our future studies. Nevertheless, this in vitro study has provided novel evidence for a greater extent of dysfunction in
Ppt1−/− glial cells than was previously anticipated and highlights the need for targeting glial cells in developing therapies for CLN1 disease. Gene therapy using newer generations of adeno-associated viruses has shown increasing efficacy, especially if these are targeted simultaneous to the brain and spinal cord [
43]. These vectors predominantly transduce neurons, and these would secret Ppt1 enzyme to cross correct Ppt1 deficient glia within the CNS and correct their defects [
10,
15,
45]. Regardless of such cross-correction, the administration of anti-inflammatory compounds to
Ppt1−/− mice provides additional benefit above gene therapy alone [
28], and greater therapeutic efficacy may yet be provided by testing other drugs of this type. In a clinical setting, such neuroimmunomodulatory or anti-inflammatory approaches may be of use either before gene therapy can be administered or as an adjunct to gene therapy.
The NCLs have been grouped together traditionally on the basis of certain broadly similar clinical and pathological themes, but as new data emerge it is becoming evident that this is an oversimplification. It is clear that cultured astrocytes and microglia generated from CLN1 (this study) and CLN3 mice [
35] are dysfunctional and exert a negative influence upon neuron health and may ultimately contribute to neurodegeneration. However, as the CLN1 data in this study reveal, the nature of these events differs markedly between these two major forms of NCL. Perhaps this is not surprising given that these two forms of NCL are caused by mutations in genes that encode such different types of proteins, one a lysosomal hydrolase (CLN1/PPT1) and the other a transmembrane protein whose function remains obscure (CLN3). Nevertheless, these findings have direct implications for our understanding of the cellular pathogenesis of these disorders and in this respect, it will be important to extend these studies to other forms of NCL.