Rho GTPases in the Amygdala-A Switch for Fears?

Fear is a fundamental evolutionary process for survival. However, excess or irrational fear hampers normal activity and leads to phobia. The amygdala is the primary brain region associated with fear learning and conditioning. There, Rho GTPases are molecular switches that act as signaling molecules for further downstream processes that modulate, among others, dendritic spine morphogenesis and thereby play a role in fear conditioning. The three main Rho GTPases-RhoA, Rac1, and Cdc42, together with their modulators, are known to be involved in many psychiatric disorders that affect the amygdala's fear conditioning mechanism. Rich2, a RhoGAP mainly for Rac1 and Cdc42, has been studied extensively in such regard. Here, we will discuss these effectors, along with Rich2, as a molecular switch for fears, especially in the amygdala. Understanding the role of Rho GTPases in fear controlling could be beneficial for the development of therapeutic strategies targeting conditions with abnormal fear/anxiety-like behaviors.

Reduced plaque size and inflammation in the APP23 mouse model for Alzheimer's disease after chronic application of polymeric nanoparticles for CNS targeted zinc delivery.

A local dyshomeostasis of zinc ions in the vicinity of amyloid aggregates has been proposed in Alzheimer's disease (AD) due to the sequestration of zinc in senile plaques. While an increase in zinc levels may promote the aggregation of amyloid beta (Aß), increased brain zinc might also be beneficial rescuing some pathological alterations caused by local zinc deficiency. For example, increased Aß degradation by metalloproteinases, and a reduction in inflammation can be hypothesized. In addition, zinc may allow a stabilization of the number of synapses in AD brains. Thus, to evaluate whether altering zinc-levels within the brain is a promising new target for the prevention and treatment of AD, we employed novel zinc loaded nanoparticles able to deliver zinc into the brain across the blood-brain barrier. We performed in vivo studies using wild type (WT) and APP23 mice to assess plaque load, inflammatory status and synapse loss. Furthermore, we performed behavioral analyses. After chronically injecting these nanoparticles for 14 days, our results show a significant reduction in plaque size and effects on the pro-inflammatory cytokines IL-6 and IL-18. On behavioral level we could not detect negative effects of increased brain zinc levels in APP23 mice and treatment with g7-NP-Zn normalized the observed hyperlocomotion of APP23 mice. Therefore, we conclude that a targeted increase in brain zinc levels may have beneficial effects in AD.

Object Phobia and Altered RhoA Signaling in Amygdala of Mice Lacking RICH2.

RICH2 knockout (RICH2 KO) mice exhibit neophobia in the novel object test. To gain further insight into their anxiety-related phenotype, we subjected these mice to additional behavioral tests to elucidate whether the behavioral abnormality in these mice is a consequence of reduced exploratory motivation, and whether the neophobia is linked specifically to objects or also present for other modalities. RICH2 KO mice engage in normal exploration in a novel environment, suggesting that the anxiety-related phenotype is not due to reduced exploratory drive. Increased fear response was not observed using novel olfactory cues, but restricted to objects. Given that the amygdala is an important brain region mediating anxiety-related behaviors and a prime target for anxiety-related therapeutics, and RICH2 is a Rho-GTPase activating protein (GAP) regulating synaptic spine plasticity via small GTPases, we analyzed spine formation, morphology and receptor composition in amygdala. We found disinhibition of RhoA in the amygdala of RICH2 KO mice, along with a decreased ability for actin polymerization and a reduction in mature spines. However, we detected increased neuronal activation in the amygdala evidenced by c-fos labeling. Thus, we conclude that despite unaltered baseline activity, RICH2 KO mice show heightened amygdala response after exposure to objects, which, however, does not result in homeostatic strengthening of excitatory synapses.

Actin-Dependent Alterations of Dendritic Spine Morphology in Shankopathies.

Shank proteins (Shank1, Shank2, and Shank3) act as scaffolding molecules in the postsynaptic density of many excitatory neurons. Mutations in SHANK genes, in particular SHANK2 and SHANK3, lead to autism spectrum disorders (ASD) in both human and mouse models. Shank3 proteins are made of several domains-the Shank/ProSAP N-terminal (SPN) domain, ankyrin repeats, SH3 domain, PDZ domain, a proline-rich region, and the sterile alpha motif (SAM) domain. Via various binding partners of these domains, Shank3 is able to bind and interact with a wide range of proteins including modulators of small GTPases such as RICH2, a RhoGAP protein, and ßPIX, a RhoGEF protein for Rac1 and Cdc42, actin binding proteins and actin modulators. Dysregulation of all isoforms of Shank proteins, but especially Shank3, leads to alterations in spine morphogenesis, shape, and activity of the synapse via altering actin dynamics. Therefore, here, we highlight the role of Shank proteins as modulators of small GTPases and, ultimately, actin dynamics, as found in multiple in vitro and in vivo models. The failure to mediate this regulatory role might present a shared mechanism in the pathophysiology of autism-associated mutations, which leads to dysregulation of spine morphogenesis and synaptic signaling.

Activity and circadian rhythm influence synaptic Shank3 protein levels in mice.

Various recent studies revealed that the proteins of the Shank family act as major scaffold organizing elements in the post-synaptic density of excitatory synapses and that their expression level is able to influence synapse formation, maturation and ultimately brain plasticity. An imbalance in Shank3 protein levels has been associated with a variety of neuropsychological and neurodegenerative disorders including autism spectrum disorders and Phelan-McDermid syndrome. Given that sleep disorders and low melatonin levels are frequently observed in autism spectrum disorders, and that circadian rhythms may be able to modulate Shank3 signaling and thereby synaptic function, here, we performed in vivo studies on CBA mice using protein biochemistry to investigate the synaptic expression levels of Shank3α during the day in different brain regions. Our results show that synaptic Shank3 protein concentrations exhibit minor oscillations during the day in hippocampal and striatal brain regions that correlate with changes in serum melatonin levels. Furthermore, as circadian rhythms are tightly connected to activity levels in mice, we increased physical activity using running wheels. The expression of Shank3α increases rapidly by induced activity in thalamus and cortex, but decreases in striatum, superimposing the circadian rhythms of different brain regions. We conclude that synaptic Shank3 proteins build highly dynamic platforms that are modulated by the light:dark cycles but even more so driven by activity. Using wild-type CBA mice, we show that Shank3 is a highly dynamic and activity-regulated protein at synapses. In the hippocampus, changes in synaptic Shank3 levels are influenced by circadian rhythm/melatonin concentration, while running activity increases and decreases levels of Shank3 in the cortex and striatum respectively.

Enlarged dendritic spines and pronounced neophobia in mice lacking the PSD protein RICH2.

BACKGROUND: The majority of neurons within the central nervous system receive their excitatory inputs via small, actin-rich protrusions called dendritic spines. Spines can undergo rapid morphological alterations according to synaptic activity. This mechanism is implicated in learning and memory formation as it is ultimately altering the number and distribution of receptors and proteins at the post-synaptic membrane, thereby regulating synaptic input. The Rho-family GTPases play an important role in regulating this spine plasticity by the interaction with cytoskeletal components and several signaling pathways within the spine compartment. Rho-GAP interacting CIP4 homologue2/RICH2 is a Rho-GAP protein regulating small GTPases and was identified as an interaction partner of the scaffolding protein SHANK3 at post-synaptic densities. RESULTS: Here, we characterize the loss of RICH2 in a novel mouse model. Our results show that RICH2 KO animals display a selective and highly significant fear of novel objects and increased stereotypic behavior as well as impairment of motor learning. We found an increase in multiple spine synapses in the hippocampus and cerebellum along with alterations in receptor composition and actin polymerization. Furthermore, we observed that the loss of RICH2 leads to a disinhibition of synaptic RAC1 in vivo. CONCLUSIONS: The results are in line with the reported role of RAC1 activity being essential for activity-dependent spine enlargement. Since SHANK3 mutations are known to be causative for neuropsychiatric diseases of the Autism Spectrum (ASD), a disintegrated SHANK3/RICH2 complex at synaptic sites might at least in part be responsible for abnormal spine formation and plasticity in ASDs.

Application of Polymeric Nanoparticles for CNS Targeted Zinc Delivery In Vivo.

A dyshomeostasis of zinc ions has been reported for many psychiatric and neurodegenerative disorders including schizophrenia, attention deficit hyperactivity disorder, depression, autism, Parkinson's and Alzheimer's disease. Furthermore, alterations in zinc-levels have been associated with seizures and traumatic brain injury. Thus, altering zinclevels within the brain is emerging as a new target for the prevention and treatment of psychiatric and neurological diseases. However, given the restriction of zinc uptake into the brain by the blood-brain barrier, methods for controlled regulation and manipulation of zinc concentrations within the brain are rare. Here, we performed in vivo studies investigating the possibility of brain targeted zinc delivery using zinc-loaded nanoparticles which are able to cross the blood-brain barrier. After injecting these nanoparticles, we analyzed the regional and time-dependent distribution of zinc and nanoparticles within the brain. Moreover, we evaluated whether the presence of zinc-loaded nanoparticles alters the expression of zinc sensitive genes and proteins such as metallothioneins and zinc transporters and quantified possible toxic effects. Our results show that zinc loaded g7 nanoparticles offer a promising approach as a novel non - invasive method to selectively enrich zinc in the brain within a small amount of time.