Proteomic analyses reveal misregulation of LIN28 expression and delayed timing of glial differentiation in human iPS cells with MECP2 loss-of-function.

Rett syndrome (RTT) is a pervasive developmental disorder caused by mutations in MECP2. Complete loss of MECP2 function in males causes congenital encephalopathy, neurodevelopmental arrest, and early lethality. Induced pluripotent stem cell (iPSC) lines from male patients harboring mutations in MECP2, along with control lines from their unaffected fathers, give us an opportunity to identify some of the earliest cellular and molecular changes associated with MECP2 loss-of-function (LOF). We differentiated iPSC-derived neural progenitor cells (NPCs) using retinoic acid (RA) and found that astrocyte differentiation is perturbed in iPSC lines derived from two different patients. Using highly stringent quantitative proteomic analyses, we found that LIN28, a gene important for cell fate regulation and developmental timing, is upregulated in mutant NPCs compared to WT controls. Overexpression of LIN28 protein in control NPCs suppressed astrocyte differentiation and reduced neuronal synapse density, whereas downregulation of LIN28 expression in mutant NPCs partially rescued this synaptic deficiency. These results indicate that the pathophysiology of RTT may be caused in part by misregulation of developmental timing in neural progenitors, and the subsequent consequences of this disruption on neuronal and glial differentiation.

Transport of a kinesin-cargo pair along microtubules into dendritic spines undergoing synaptic plasticity.

Synaptic plasticity often involves changes in the structure and composition of dendritic spines. Vesicular cargos and organelles enter spines either by exocytosing in the dendrite shaft and diffusing into spines or through a kinesin to myosin hand-off at the base of spines. Here we present evidence for microtubule (MT)-based targeting of a specific motor/cargo pair directly into hippocampal dendritic spines. During transient MT polymerization into spines, the kinesin KIF1A and an associated cargo, synaptotagmin-IV (syt-IV), are trafficked in unison along MTs into spines. This trafficking into selected spines is activity-dependent and results in exocytosis of syt-IV-containing vesicles in the spine head. Surprisingly, knockdown of KIF1A causes frequent fusion of syt-IV-containing vesicles throughout the dendritic shaft and diffusion into spines. Taken together, these findings suggest a mechanism for targeting dendritic cargo directly into spines during synaptic plasticity and indicate that MT-bound kinesins prevent unregulated fusion by sequestering vesicular cargo to MTs.

Synaptic regulation of microtubule dynamics in dendritic spines by calcium, F-actin, and drebrin.

Dendritic spines are actin-rich compartments that protrude from the microtubule-rich dendritic shafts of principal neurons. Spines contain receptors and postsynaptic machinery for receiving the majority of glutamatergic inputs. Recent studies have shown that microtubules polymerize from dendritic shafts into spines and that signaling through synaptic NMDA receptors regulates this process. However, the mechanisms regulating microtubule dynamics in dendrites and spines remain unclear. Here we show that in hippocampal neurons from male and female mice, the majority of microtubules enter spines from highly localized sites at the base of spines. These entries occur in response to synapse-specific calcium transients that promote microtubule entry into active spines. We further document that spine calcium transients promote local actin polymerization, and that F-actin is both necessary and sufficient for microtubule entry. Finally, we show that drebrin, a protein known to mediate interactions between F-actin and microtubules, acts as a positive regulator of microtubule entry into spines. Together these results establish for the first time the essential mechanisms regulating microtubule entry into spines and contribute importantly to our understanding of the role of microtubules in synaptic function and plasticity.

Dynamic microtubules promote synaptic NMDA receptor-dependent spine enlargement.

Most excitatory synaptic terminals in the brain impinge on dendritic spines. We and others have recently shown that dynamic microtubules (MTs) enter spines from the dendritic shaft. However, a direct role for MTs in long-lasting spine plasticity has yet to be demonstrated and it remains unclear whether MT-spine invasions are directly influenced by synaptic activity. Lasting changes in spine morphology and synaptic strength can be triggered by activation of synaptic NMDA receptors (NMDARs) and are associated with learning and memory processes. To determine whether MTs are involved in NMDAR-dependent spine plasticity, we imaged MT dynamics and spine morphology in live mouse hippocampal pyramidal neurons before and after acute activation of synaptic NMDARs. Synaptic NMDAR activation promoted MT-spine invasions and lasting increases in spine size, with invaded spines exhibiting significantly faster and more growth than non-invaded spines. Even individual MT invasions triggered rapid increases in spine size that persisted longer following NMDAR activation. Inhibition of either NMDARs or dynamic MTs blocked NMDAR-dependent spine growth. Together these results demonstrate for the first time that MT-spine invasions are positively regulated by signaling through synaptic NMDARs, and contribute to long-lasting structural changes in targeted spines.

BDNF-induced increase of PSD-95 in dendritic spines requires dynamic microtubule invasions.

Microtubules (MTs) are capable of entering dendritic spines in mature hippocampal neurons through dynamic polymerization. Although these MT invasions are directly associated with neuronal activity, their function remains unknown. Here we demonstrate in mouse hippocampal neurons that MT entries into spines regulate the increase in postsynaptic density-95 (PSD-95) protein after brain-derived neurotrophic factor (BDNF) treatment. Using multiwavelength total internal reflectance fluorescence microscopy, we show that BDNF prolonged the average MT dwell time in spines and that this effect was dependent on TrkB receptor activation. Further examination revealed that peaks of MT polymerization into spines corresponded to rapid PSD-95 increases in the spine head. Over time, spines targeted by MTs after BDNF application, but not before, showed a robust increase in PSD-95. Conversely, spines completely devoid of MT invasions showed no significant change in the level of PSD-95. Pharmacological inhibition of MT dynamics abolished the BDNF-induced increase in PSD-95. Together, these results support the hypothesis that the well known increase in PSD-95 within spines after BDNF treatment is dependent on MT invasions of dendritic spines. Thus, our study provides a direct link between dynamic MTs and the postsynaptic structure, and provides a functional role for MT invasion of dendritic spines.

The dynamic cytoskeleton: backbone of dendritic spine plasticity.

Dendritic spines are small actin-rich protrusions on the surface of dendrites whose morphological and molecular plasticity play key roles in learning and memory. Both the form and function of spines are critically dependent on the actin cytoskeleton. However, new research, using electron microscopy and live-cell super-resolution microscopy indicates that the actin cytoskeleton is more complex and dynamic than originally thought. Also, exciting recent studies from several labs indicate that microtubules, once thought to be restricted to the dendrite shaft, can make excursions into the most distal regions of dendritic spines. Moreover, microtubule invasions of spines appear to be associated with changes in synaptic activity. Thus, it is likely that dynamic interactions between microtubules and actin filaments within dendritic spines play important roles in dendritic spine plasticity.

Activity-dependent dynamic microtubule invasion of dendritic spines.

Dendritic spines are the primary sites of contact with presynaptic axons on excitatory hippocampal and cortical neurons. During development and plasticity spines undergo marked changes in structure that directly affect the functional communication between neurons. Elucidating the cytoskeletal events that induce these structural changes is fundamental to understanding synaptic biology. Actin plays a central role in the spine cytoskeleton, however the role of microtubules in spine function has been studied little. Although microtubules have a prominent role in transporting material throughout the dendrite that is destined for spines, they are not thought to directly influence spine structure or function. Using total internal reflectance fluorescent microscopy we discovered that microtubules rapidly invade dendritic protrusions of mature CNS neurons (up to 63 d in vitro), occasionally being associated with marked changes in spine morphology in the form of transient spine head protrusions (tSHPs). Two microtubules can occupy a spine simultaneously and microtubule targeting can occur from both the proximal and distal dendrite. A small percentage of spines are targeted at a time and all targeting events are transient, averaging only a few minutes. Nevertheless, over time many spines on a dendrite are targeted by microtubules. Importantly, we show that increasing neuronal activity enhances both the number of spines invaded by microtubules and the duration of these invasions. This study provides new insight into the dynamics of the neuronal cytoskeleton in mature CNS neurons and suggests that microtubules play an important, direct role in spine morphology and function.