A mutation in either of two genes (FLNA
has been shown to cause PH in humans (27
). While these two genes appear quite disparate functionally, the similarities in the radiographic characteristics of bilateral heterotopic nodules, between cases of PH arising from FLNA
mutation and cases arising from ARFGEF2
mutation, suggest that the two genes have a common final effector, in giving rise to aberrant cortical development. Through examination of post-mortem human PH brains (including a familial male case of PH due to a FLNA
mutation), we showed that the vast majority of neurons migrated into the cerebral cortex. Detailed examination of the ependymal lining of the heterotopic nodules revealed a significant disruption in the integrity of the neuroependyma. Experimental disruption of FlnA signaling in mice, both in vivo
and in vitro
, resulted in a failure to initiate migration, an altered integrity of the ependymal lining and a loss of cell adhesion and cell–cell contacts. Inhibition of Arfgef2
/Big2 induced by BFA caused a similar disruption in the neuroepithelial lining and led to formation of periventricular nodules in mice. Finally, we described the formation of nodules in the hyh
mouse: this mouse's mutation in the Napa
gene disrupts SNARE receptor (SNARE)-mediated vesicle transport and membrane fusion. Because alpha-SNAP is a protein involved in vesicle fusion, it should be widely expressed in the body. However, evidence indicates that it is preferentially expressed in the CNS and at early developmental stages (56
); within the CNS, alpha-SNAP is most highly expressed in the VZ and SVZ (present report). This expression pattern would explain why the alpha-SNAP mutation is principally expressed spatially in these zones, and at certain stages of development. Overall, our results suggest that PH, while linked to aberrations in neuronal motility, also can arise from a disruption in the neuroependyma, where loss of FlnA, Arfgef2
/Big2 and Napa
/alpha-SNAP functions alter vesicle trafficking and the adhesion of neural progenitors.
Genetic studies and clinical observations associated with X-linked PH suggest that the role of FLNA in cell motility is not entirely responsible for periventricular nodule formation. For example, random X-inactivation would imply that ~50% of the neurons born would not express the normal filamin protein. However, in most reported PH cases and in the post-mortem tissues examined here, the overlying cortex is relatively normal, and only a small proportion of the neurons constitute the nodules (42
). Moreover, recent genetic analysis has identified FLNA
mutations in several male patients, many of whom were not somatic mosaics for FLNA
mutations (as was the case for the 37-week GA male included in this study), implying that—despite the fact that all neurons bear the FLNA
mutation—the neurons either migrate completely into the cortex or do not migrate at all (but never halfway) (28
). Furthermore, autosomal recessive mutations in ARFGEF2
result in PH with radiographic findings virtually identical to these seen in PH due to a FLNA
mutation, except for the presence of the severe microcephaly associated with ARFGEF2
). In ARFGEF
-related PH, all of the neurons necessarily harbor homozygous mutations in ARFGEF2
, but still form heterotopic nodules and a relatively normal-appearing cortex, suggesting that the periventricular nodules are unlikely to be due to motility impairments alone. Finally, the recessive nature of the disorder suggests that PH is not a cell-autonomous process, given that many neurons migrate appropriately even while others do not.
Several aspects of our current findings suggest that PH does not arise solely from a cell-autonomous motility defect. Most neurons actually migrate into the developing cerebral cortex and take up their proper final positions in the appropriate cortical layers, as evidenced by the precise lamination seen with FOXP1 and CUX1 immunostaining. The lack of FOXP1 neuronal expression in the nodules suggests that the earliest-born neurons were able to migrate from the VZ and into the appropriate lower layers of the cerebral cortex (as indicated by the expression of FOXP1 in the deep layers of the cortex). The increased proportion of CUX1-positive neurons in the nodules, however, would argue that the later-born neurons were the ones that were predominantly affected. This preponderance of later-born neurons in the nodules is not necessarily consistent with PH's being a cell-autonomous cell motility problem; if that were the case, the migration of all neuronal populations would be expected to adversely affected. Rather, a loss of ependymal integrity, which occurs later in development, would be a more likely reason why later-born neurons are affected. GABAergic neurons that arise from the ganglionic eminences and undergo long-distance migration to the VZ are found in the nodules suggesting that some migration of this neuronal population occurred appropriately. Finally, recent studies show that the mutant FlnA
mouse, while embryonic lethal, does not impair neuronal migration during embryogenesis (35
). Taken in sum, our data are most consistent with the idea that FLNA
mutations cause PH, secondary to impairments in neuronal motility (cell-autonomous), but also PH can arise from a disruption in neural progenitors along the VZ, a disruption that influences the migration of later-born neurons (non-cell-autonomous).
Previous studies have suggested that PH arises from an impairment of the initial migration of neurons. Inhibition of FlnA signaling by dominant-negative FlnA transfection was shown, in prior work, as well as here, to disrupt the initial migration and the morphology of migratory neuroblasts in the SVZ (48
). Furthermore, FilaminA-interacting protein (FILIP) has been suggested to be important, in allowing neurons to exit the VZ (48
). Such observations, however, do not necessarily explain the preponderance of later-born neurons in the nodules, unless both FILIP and FLNA are more critical for the decision by later-born migratory neurons to exit the SVZ. Later-born post-mitotic neurons do migrate longer distances and must migrate past earlier-born neurons as they enter the more superficial layers of cortex, suggesting that additional cues are required by the later-born neurons. Our expression data show that both FlnA and Big2 proteins are highly expressed in, and strongly restricted to the neuroependymal lining; they are not highly expressed in the SVZ. Loss of FLNA function has additional effects; it can cause aortic rupture, gut dysmotility and increased skin elasticity (27
), with high FLNA expression restricted to the lining of the blood vessel, intestines and skin, respectively (Supplementary Material, Fig. S8
). Defects in the linings of these organs could in part account for the above phenotypes.
Several observations suggest that PH does not result from an over-production of neurons. The brains of individuals with PH that arises from ARFGEF2
mutations are microcephalic, and Big2 inhibition in vitro
leads to diminished proliferation of neurons (37
). A recent case study of PH suggested that microcephaly in males can be associated with FLNA
). Neural precursors (neurospheres) and fibroblasts obtained from Case 3 failed to demonstrate any increase in proliferation (data not shown). Similarly, BrdU labeling of proliferating cells in neurospheres obtained from hyh
mutant mice showed no difference from wild-type neurospheres (data not shown). The hyh
mice showed a progressive loss of proliferative progenitor cells during development (57
). Direct inhibition of FlnA signaling in HEK293 cells by dominant-negative over-expression did not produce any increase in cell number (data not shown). Taken together, these observations suggest that nodule formation in the PH brain is not a direct consequence of over-proliferation of neurons along the VZ during cerebral cortical development. However, studies have suggested that endothelial cells can stimulate their own self renewal, and can expand neurogenesis in NSCs (65
). Thus, it remains to be determined whether the loss of blood brain barrier integrity along the VZ, or the endothelial vasculature, contributes to an increase in neural progenitor proliferation, and periventricular nodule formation.
Disruption of the neuroepithelial lining, in giving rise to PH, mirrors the loss of structural integrity along the pial surface of the brain that produces cobblestone lissencephaly (66
). In cobblestone lissencephaly, neurons have no inherent motility defect; rather, they migrate beyond the marginal zone into the leptomeninges and through the external basement membrane during cortical development. The loss of integrity of the molecular layer leads to nodules composed of neurons, on the outermost surface of the brain (thought to resemble cobblestones). Four genes, fukutin
, have been associated with cobblestone lissencephaly (69
); all are implicated in the glycosylation of dystroglycans. Hypoglycosylation of dystroglycan abolishes binding activity for such ligands as laminin, neurexin and agrin, and thereby compromises the integrity of the dystrophin-associated extracellular matrix adhesion complex (72
). In a fashion akin to neuronal migration, neural progenitors perform interkinetic nuclear migration in the VZ, with cells in synthesis (S) phase positioned in the upper half of the epithelium away from the ventricular surface (73
). Cells transitioning from S to growth (G) 2 phase have nuclei that translocate in the cell toward the ventricular surface, and cells in mitosis (M) lie adjacent to the ventricular surface. Following M phase, cells re-entering G1 phase translocate away from the neuroepithelium, while post-mitotic neurons generally migrate toward the pial surface and cortical plate. Loss of the neuroepithelial integrity along the ventricular surface could conceivably disrupt cells exiting M phase, causing them to ‘over-migrate’ or to protrude into the ventricular lumen, giving rise to periventricular nodules. This possibility would be consistent with the observed loss of neuroependymal integrity in the human pathological cases of PH, in the hyh
mutant mouse and in the mouse models in which inhibition of FlnA and Big2 occurred. Thus, in some ways, PH reflects a pathological process similar to that seen in cobblestone lissencephaly; the difference is merely that in PH the inner neuroependymal lining is affected, rather than outer surface of the brain.
Several lines of evidence suggest that PH results from a disruption of vesicle trafficking. First, a number of peripheral membrane proteins, including FLNA, associate dynamically with Golgi membranes during the budding and trafficking of transport vesicles in eukaryotic cells (50
). FLNA has been shown to regulate furin sorting in the trans
-Golgi network/endosomal system (51
). Thus, while FLNA regulation of actin may directly alter the cytoskeleton required for migration, an additional role for FLNA, in vesicle trafficking, would not be surprising, given that the actin cytoskeleton is required for vesicle budding and vesicle movement (76
). Second, other genes implicated in periventricular nodule formation appear to be involved in vesicle trafficking. ARFGEF2 regulates the ARFs, which bind to vesicle coat proteins and adaptors. Similarly, the studies reported in the present paper suggest that mutations in Napa
also result in PH in the hyh
mouse. Alpha-SNAP is involved in SNARE-mediated vesicle fusion in many cellular contexts (79
). Finally, Mekk4, while it has not been directly linked to vesicle trafficking, has been shown to interact with FlnA, and to regulate FlnA expression (86
). It remains to be seen whether each of these genes interact directly within the same pathway.
Once we better understand the functions of the genes that regulate the interaction between the actin cytoskeleton and Golgi apparatus (and consequently vesicle transport), during brain development, we will begin to start to form a clearer picture of the causal mechanisms of human PH, and the associated CNS phenotype. Heterotopic nodules are the primary feature seen in this disorder, and disruption in the transport or recycling of adherens junction molecules could explain a breakdown in the neuroepithelial lining. PH, however, is also associated with a thinning of the cortex and in extreme cases, microcephaly. Vesicle transport (via the recycling endosome) is responsible for the delivery of specific lipids and proteins to the cleavage furrow, and is crucial for cell abscission. Disruption of this process would explain the altered rates of symmetric and asymmetric division that lead to changes in neural precursor fate and a thinner cortex in PH. Finally, humans with PH show an elevated incidence of dyslexia and psychiatric disorders (80
), suggesting problems with synaptic connectivity. Impairments in actin-based vesicle trafficking could also disrupt these contacts; disruption of both FLNA and Big2 has already been shown to disrupt recycling of surface receptors and neurite extension, necessary for dendritic arborization. Further studies are needed to determine whether this common function in vesicle trafficking leads to the multiple observed phenotypes in PH.