Serena M. Dudek

Cortico-striatal synaptic defects and OCD-like behaviours in Sapap3-mutant mice.

Obsessive-compulsive disorder (OCD) is an anxiety-spectrum disorder characterized by persistent intrusive thoughts (obsessions) and repetitive actions (compulsions). Dysfunction of cortico-striato-thalamo-cortical circuitry is implicated in OCD, although the underlying pathogenic mechanisms are unknown. SAP90/PSD95-associated protein 3 (SAPAP3; also known as DLGAP3) is a postsynaptic scaffolding protein at excitatory synapses that is highly expressed in the striatum. Here we show that mice with genetic deletion of Sapap3 exhibit increased anxiety and compulsive grooming behaviour leading to facial hair loss and skin lesions; both behaviours are alleviated by a selective serotonin reuptake inhibitor. Electrophysiological, structural and biochemical studies of Sapap3-mutant mice reveal defects in cortico-striatal synapses. Furthermore, lentiviral-mediated selective expression of Sapap3 in the striatum rescues the synaptic and behavioural defects of Sapap3-mutant mice. These findings demonstrate a critical role for SAPAP3 at cortico-striatal synapses and emphasize the importance of cortico-striatal circuitry in OCD-like behaviours.

Synapse elimination accompanies functional plasticity in hippocampal neurons

A critical component of nervous system development is synapse elimination during early postnatal life, a process known to depend on neuronal activity. Changes in synaptic strength in the form of long-term potentiation (LTP) and long-term depression (LTD) correlate with dendritic spine enlargement or shrinkage, respectively, but whether LTD can lead to an actual separation of the synaptic structures when the spine shrinks or is lost remains unknown. Here, we addressed this issue by using concurrent imaging and electrophysiological recording of live synapses. Slices of rat hippocampus were cultured on multielectrode arrays, and the neurons were labeled with genes encoding red or green fluorescent proteins to visualize presynaptic and postsynaptic neuronal processes, respectively. LTD-inducing stimulation led to a reduction in the synaptic green and red colocalization, and, in many cases, it induced a complete separation of the presynaptic bouton from the dendritic spine. This type of synapse loss was associated with smaller initial spine size and greater synaptic depression but not spine shrinkage during LTD. All cases of synapse separation were observed without an accompanying loss of the spine during this period. We suggest that repeated low-frequency stimulation simultaneous with LTD induction is capable of restructuring synaptic contacts. Future work with this model will be able to provide critical insight into the molecular mechanisms of activity- and experience-dependent refinement of brain circuitry during development.

Developmental Down-Regulation of LTD in Cortical Layer IV and Its Independence of Modulation by Inhibition

For in vitro LTD to remain viable as a model for synaptic weakening in visual cortical plasticity, it is crucial that it display a critical period for its induction within layer IV. A complicating factor, however, is that LTD in layer IV is modulated by inhibitory postsynaptic potentials (IPSPs); postsynaptic responses characterized as containing IPSPs do not depress in response to 1 Hz afferent stimulation. By blocking IPSPs intracellularly, we find that the ability to induce LTD in layer IV neurons is restored in juvenile, but not in mature animals. This developmental down-regulation of LTD induction is specific for layer IV when compared with LTD induction in layers II/III. These data are consistent with the hypothesis that an LTD-like phenomenon is involved in critical period plasticity and is apparently independent of developmental changes in inhibitory circuitry.

Transglutaminase facilitates the formation of polymers of the β-amyloid peptide

One of the major pathological characteristics of Alzheimers disease is the increased number of amyloid-containing senile plaques within the brain. The dense cores of these plaques are composed primarily of highly insoluble aggregates of a 39–43-residue peptide referred to as the β-amyloid peptide (βA). The mechanisms by which these insoluble extracellular deposits of βA are formed remain unknown. In this study, the cross-linking of βA by the calcium-dependent enzyme, transglutaminase was examined. Transglutaminases are a family of enzymes which are found in brain, and catalyse the cross-linking of specific proteins into insoluble polymers. Synthetic βA (1–40) was readily cross-linked by transglutaminase, forming multimers in a time-dependent fashion. Furthermore, a second peptide with a substitution similar to that in the Dutch-type hereditary amyloidosis mutation (Glu22 to Gln) was also found to be a substrate fro transglutaminase. Since transglutaminase covalently cross-links proteins through glutamine residues, it is suggested that transglutaminase contributes to amyloid deposition in Dutch-type hereditary amyloidosis, and possibl Alzheimers disease.

Somatic action potentials are sufficient for late-phase LTP-related cell signaling

A question of critical importance confronting neuroscientists today is how biochemical signals initiated at a synapse are conveyed to the nucleus. This problem is particularly relevant to the generation of the late phases of long-term potentiation (LTP). Here we provide evidence that some signaling pathways previously associated with late-LTP can be activated in hippocampal CA1 neurons without synaptic activity; somatic action potentials, induced by backfiring the cells, were found to be sufficient for phosphorylation of extracellular signal-regulated kinase-1/2 and cAMP response element-binding protein, as well as for induction of zif268. Furthermore, such antidromic stimulation was adequate to rescue “tagged” synapses (early-LTP) from decay. These results show that a synapse-to-nucleus signal is not necessary for late-phase LTP-associated signaling cascades in the regulation of gene expression.

Transglutaminase catalyzes the formation of sodium dodecyl sulfate-insoluble, Alz-50-reactive polymers of τ

Abstract: Paired helical filaments, a constituent of neurofibrillary tangles in Alzheimers disease, consist primarily of the microtu‐bule‐associated protein τ. However, the process by which the detergent‐insoluble filaments of the neurofibrillary tangles are formed from soluble τ remains unknown. Here, we present a potential mechanism for the abnormal aggregation of τ in Alzheimers disease: the covalent cross‐linking of τ by the enzyme transglutaminase. Macromolecular complexes of τ, formed in the presence of transglutaminase, were found to be insoluble in ionic detergent, β‐mercaptoethanol, guanidine‐HCI, and urea and, furthermore, demonstrated an increased immunoreactivity with the monoclonal antibody Alz‐50. Electron microscopic studies revealed that τ cross‐linked by transglutaminase has a defined filamentous structure. These results indicate that transglutaminase, the activity of which has been shown to increase during programmed cell death, may play a role in the formation of pathology associated with Alzheimers disease.

Rapid activity-induced transcription of Arc and other IEGs relies on poised RNA polymerase II

Transcription of immediate early genes (IEGs) in neurons is highly sensitive to neuronal activity, but the mechanism underlying these early transcription events is largely unknown. We found that several IEGs, such as Arc (also known as Arg3.1), are poised for near-instantaneous transcription by the stalling of RNA polymerase II (Pol II) just downstream of the transcription start site in rat neurons. Depletion through RNA interference of negative elongation factor, a mediator of Pol II stalling, reduced the Pol II occupancy of the Arc promoter and compromised the rapid induction of Arc and other IEGs. In contrast, reduction of Pol II stalling did not prevent transcription of IEGs that were expressed later and largely lacked promoter-proximal Pol II stalling. Together, our data strongly indicate that the rapid induction of neuronal IEGs requires poised Pol II and suggest a role for this mechanism in a wide variety of transcription-dependent processes, including learning and memory.

RGS14 is a natural suppressor of both synaptic plasticity in CA2 neurons and hippocampal-based learning and memory

Learning and memory have been closely linked to strengthening of synaptic connections between neurons (i.e., synaptic plasticity) within the dentate gyrus (DG)–CA3–CA1 trisynaptic circuit of the hippocampus. Conspicuously absent from this circuit is area CA2, an intervening hippocampal region that is poorly understood. Schaffer collateral synapses on CA2 neurons are distinct from those on other hippocampal neurons in that they exhibit a perplexing lack of synaptic long-term potentiation (LTP). Here we demonstrate that the signaling protein RGS14 is highly enriched in CA2 pyramidal neurons and plays a role in suppression of both synaptic plasticity at these synapses and hippocampal-based learning and memory. RGS14 is a scaffolding protein that integrates G protein and H-Ras/ERK/MAP kinase signaling pathways, thereby making it well positioned to suppress plasticity in CA2 neurons. Supporting this idea, deletion of exons 2–7 of the RGS14 gene yields mice that lack RGS14 (RGS14-KO) and now express robust LTP at glutamatergic synapses in CA2 neurons with no impact on synaptic plasticity in CA1 neurons. Treatment of RGS14-deficient CA2 neurons with a specific MEK inhibitor blocked this LTP, suggesting a role for ERK/MAP kinase signaling pathways in this process. When tested behaviorally, RGS14-KO mice exhibited marked enhancement in spatial learning and in object recognition memory compared with their wild-type littermates, but showed no differences in their performance on tests of nonhippocampal-dependent behaviors. These results demonstrate that RGS14 is a key regulator of signaling pathways linking synaptic plasticity in CA2 pyramidal neurons to hippocampal-based learning and memory but distinct from the canonical DG–CA3–CA1 circuit.

Synaptic Plasticity (and the Lack Thereof) in Hippocampal CA2 Neurons

The hippocampus is critical for some forms of memory and spatial navigation, but previous research has mostly neglected the CA2, a unique region situated between CA3 and CA1. Here, we show that CA2 pyramidal neurons have distinctive physiological characteristics that include an unprecedented synaptic stability. Although basal synaptic currents in CA1 and CA2 are quite similar, synaptic plasticity including long-term potentiation and long-term depression is absent or less likely to be induced with conventional methods of stimulation in CA2. We also find that CA2 neurons have larger leak currents and more negative resting membrane potentials than CA1 neurons, and consequently, more current is required for action potential generation in CA2 neurons. These data suggest that the molecular “conspiracy against plasticity” in CA2 makes it functionally distinct from the other hippocampal CA regions. This work provides critical insight into hippocampal function and may lead to an understanding of the resistance of CA2 to damage from disease, trauma, and hypoxia.

WRP/srGAP3 Facilitates the Initiation of Spine Development by an Inverse F-BAR Domain, and Its Loss Impairs Long-Term Memory

The WAVE-associated Rac GAP, WRP, is thought to regulate key aspects of synapse development and function and may be linked to mental retardation in humans. WRP contains a newly described inverse F-BAR (IF-BAR) domain of unknown function. Our studies show that this domain senses/facilitates outward protrusions analogous to filopodia and that the molecular basis for this is likely explained by a convex lipid-binding surface on the WRP IF-BAR domain. In dendrites the IF-BAR domain of WRP forms a bud on the shaft from which precursors to spines emerge. Loss of WRP in vivo and in vitro results in reduced density of spines. In vivo this is primarily a loss of mushroom-shaped spines. Developmentally, WRP function is critical at the onset of spinogenesis, when dendritic filopodia are prevalent. Finally, because WRP is implicated in mental retardation, behaviors of WRP heterozygous and null mice have been evaluated. Results from these studies confirm that loss of WRP is linked to impaired learning and memory.