Marc Flajolet

Dichotomous dopaminergic control of striatal synaptic plasticity.

At synapses between cortical pyramidal neurons and principal striatal medium spiny neurons (MSNs), postsynaptic D1 and D2 dopamine (DA) receptors are postulated to be necessary for the induction of long-term potentiation and depression, respectively—forms of plasticity thought to underlie associative learning. Because these receptors are restricted to two distinct MSN populations, this postulate demands that synaptic plasticity be unidirectional in each cell type. Using brain slices from DA receptor transgenic mice, we show that this is not the case. Rather, DA plays complementary roles in these two types of MSN to ensure that synaptic plasticity is bidirectional and Hebbian. In models of Parkinsons disease, this system is thrown out of balance, leading to unidirectional changes in plasticity that could underlie network pathology and symptoms.

Gamma-secretase activating protein is a therapeutic target for Alzheimer’s disease

Accumulation of neurotoxic amyloid-β is a major hallmark of Alzheimer’s disease. Formation of amyloid-β is catalysed by γ-secretase, a protease with numerous substrates. Little is known about the molecular mechanisms that confer substrate specificity on this potentially promiscuous enzyme. Knowledge of the mechanisms underlying its selectivity is critical for the development of clinically effective γ-secretase inhibitors that can reduce amyloid-β formation without impairing cleavage of other γ-secretase substrates, especially Notch, which is essential for normal biological functions. Here we report the discovery of a novel γ-secretase activating protein (GSAP) that drastically and selectively increases amyloid-β production through a mechanism involving its interactions with both γ-secretase and its substrate, the amyloid precursor protein carboxy-terminal fragment (APP-CTF). GSAP does not interact with Notch, nor does it affect its cleavage. Recombinant GSAP stimulates amyloid-β production in vitro. Reducing GSAP concentrations in cell lines decreases amyloid-β concentrations. Knockdown of GSAP in a mouse model of Alzheimer’s disease reduces levels of amyloid-β and plaque development. GSAP represents a type of γ-secretase regulator that directs enzyme specificity by interacting with a specific substrate. We demonstrate that imatinib, an anticancer drug previously found to inhibit amyloid-β formation without affecting Notch cleavage, achieves its amyloid-β-lowering effect by preventing GSAP interaction with the γ-secretase substrate, APP-CTF. Thus, GSAP can serve as an amyloid-β-lowering therapeutic target without affecting other key functions of γ-secretase.

Regulation of Alzheimer's disease amyloid-β formation by casein kinase I

Alzheimers disease (AD) is associated with accumulation of the neurotoxic peptide amyloid-β (Aβ), which is produced by sequential cleavage of amyloid precursor protein (APP) by the aspartyl protease β-secretase and the presenilin-dependent protease γ-secretase. An increase of casein kinase 1 (CK1) expression has been described in the human AD brain. We show, by using in silico analysis, that APP, β-secretase, and γ-secretase subunits contain, in their intracellular regions, multiple CK1 consensus phosphorylation sites, many of which are conserved among human, rat, and mouse species. Overexpression of constitutively active CK1ε, one of the CK1 isoforms expressed in brain, leads to an increase in Aβ peptide production. Conversely, three structurally dissimilar CK1-specific inhibitors significantly reduced endogenous Aβ peptide production. By using mammalian cells expressing the β C-terminal fragment of APP, it was possible to demonstrate that CK1 inhibitors act at the level of γ-secretase cleavage. Importantly, Notch cleavage was not affected. Our results indicate that CK1 represents a therapeutic target for prevention of Aβ formation in AD.

FGF acts as a co-transmitter through adenosine A2A receptor to regulate synaptic plasticity

Abnormalities of striatal function have been implicated in several major neurological and psychiatric disorders, including Parkinsons disease, schizophrenia and depression. Adenosine, via activation of A2A receptors, antagonizes dopamine signaling at D2 receptors and A2A receptor antagonists have been tested as therapeutic agents for Parkinsons disease. We found a direct physical interaction between the G protein–coupled A2A receptor (A2AR) and the receptor tyrosine kinase fibroblast growth factor receptor (FGFR). Concomitant activation of these two classes of receptors, but not individual activation of either one alone, caused a robust activation of the MAPK/ERK pathway, differentiation and neurite extension of PC12 cells, spine morphogenesis in primary neuronal cultures, and cortico-striatal plasticity that was induced by a previously unknown A2AR/FGFR-dependent mechanism. The discovery of a direct physical interaction between the A2A and FGF receptors and the robust physiological consequences of this association shed light on the mechanism underlying FGF functions as a co-transmitter and open new avenues for therapeutic interventions.

Roscovitine-derived, dual-specificity inhibitors of cyclin-dependent kinases and casein kinases 1.

Cyclin-dependent kinases (CDKs) and casein kinases 1 (CK1) are involved in the two key molecular features of Alzheimers disease, production of amyloid-beta peptides (extracellular plaques) and hyper-phosphorylation of Tau (intracellular neurofibrillary tangles). A series of 2,6,9-trisubstituted purines, structurally related to the CDK inhibitor roscovitine, have been synthesized. They mainly differ by the substituent on the C-6 position. These compounds were screened for kinase inhibitory activities and antiproliferative effects. Several biaryl derivatives displayed potent inhibition of both CDKs and CK1. In particular, derivative 13a was a potent inhibitor of CDK1/cyclin B (IC 50: 220 nM), CDK5/p25 (IC 50: 80 nM), and CK1 (IC 50: 14 nM). Modeling of these molecules into the ATP-binding pocket of CK1delta provided a rationale for the increased selectivity toward this kinase. 13a was able to prevent the CK1-dependent production of amyloid-beta in a cell model. CDK/CK1 dual-specificity inhibitors may have important applications in Alzheimers disease and cancers.

A small-molecule enhancer of autophagy decreases levels of Aβ and APP-CTF via Atg5-dependent autophagy pathway

The hallmarks of Alzheimers disease are the aggregates of amyloid‐β (Aβ) peptide and tau protein. Autophagy is one major cellular pathway leading to the removal of aggregated proteins. We examined the possibility of inducing autophagy to reduce Aβ peptide and the amyloid precursor protein (APP)‐derived fragment APP‐CTF levels in cell lines and primary neuronal cultures. We found that induction of autophagy either by small‐molecule enhancers of rapa‐mycin (SMER)28, a small‐molecule enhancer of autophagy, or following starvation greatly decreased the levels of Aβ peptide (apparent EC50 of ~10 µM) and APP‐CTF (apparent EC50 of ~20 µM) in a γ‐secretase‐independent manner. Pharmacological inhibition of autophagy led to a significant accumulation of Aβ peptide and APP‐CTF and diminished the effect of SMER28. Three essential components of the autophagic pathway, autophagy‐related protein (Atg)5, Beclin1, and Ulk1, were shown to be involved in the degradation of Aβ and APP‐CTF, and Atg5 was necessary for the effect of SMER28. In addition, the autophagic marker light chain 3‐II cocompartmentalized with APP‐CTF. These results support the involvement of autophagy in the clearance of Aβ and APP‐CTF. We therefore propose that small molecule enhancers of autophagy, such as SMER28, may have therapeutic potential for the treatment of Alzheimers disease and other proteinopathies.—Tian, Y., Bustos, V., Flajolet, M., Greengard, P. A small‐molecule enhancer of autophagy decreases levels of Aβ and APP‐CTF via Atg5‐dependent autophagy pathway. FASEB J. 25, 1934‐1942 (2011). www.fasebj.org

Role of p11 in Cellular and Behavioral Effects of 5-HT4 Receptor Stimulation

p11 (S100A10), a member of a large family of S100 proteins, interacts with serotonin receptor 1B (5-HTR1B), modulates 5-HT1B receptor signal transduction, and is required for antidepressant responses to activation of this receptor. In the current study, we investigated the specificity of the interaction between 5-HTR1B and p11 by screening brain-expressed S100 proteins against serotonin and noradrenergic receptors. The data indicate that p11 is unique among its family members for its interactions with defined serotonin receptors. We identify a novel p11-interacting receptor (5-HTR4) and characterize the interaction between p11 and 5-HTR4, demonstrating that (1) p11 and 5-HTR4 mRNA and protein are coexpressed in brain regions that are relevant for major depression, (2) p11 increases 5-HTR4 surface expression and facilitates 5-HTR4 signaling, and (3) p11 is required for the behavioral antidepressant responses to 5-HTR4 stimulation in vivo. The essential role played by p11 in modulating signaling through 5-HT4 as well as 5-HT1B receptors supports the concept that this protein may be a key determinant of vulnerability to depression.

Advances in the pharmacological treatment of Parkinson's disease: targeting neurotransmitter systems

For several decades, the dopamine precursor levodopa has been the primary therapy for Parkinsons disease (PD). However, not all of the motor and non-motor features of PD can be attributed solely to dopaminergic dysfunction. Recent clinical and preclinical advances provide a basis for the identification of additional innovative therapeutic options to improve the management of the disease. Novel pharmacological strategies must be optimized for PD by: (i) targeting disturbances of the serotonergic, noradrenergic, glutamatergic, GABAergic, and cholinergic systems in addition to the dopaminergic system, and (ii) characterizing alterations in the levels of neurotransmitter receptors and transporters that are associated with the various manifestations of the disease.

Adaptor complex AP2/PICALM, through interaction with LC3, targets Alzheimer's APP-CTF for terminal degradation via autophagy

Significance β-Amyloid aggregates are often found in the brains of Alzheimer’s patients. We have previously reported that β-amyloid levels can be down-regulated through activation of autophagy, a process involving the degradation of unnecessary or harmful proteins. However, the underlying mechanism of β-amyloid autophagy-mediated degradation is unknown. Here we demonstrate that the complex adaptor protein 2/phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (AP2/PICALM), via an interaction with a β-amyloid precursor, bridges β-amyloid degradation and autophagy. This work reveals mechanistic steps for the targeting of the β-amyloid precursor for degradation via autophagy and supports a genome-wide association study identifying PICALM as a risk factor for Alzheimer’s disease (AD). Altogether, these findings support the notion that activating autophagy is a valid approach for the AD field, which urgently needs novel therapeutic strategies. The hallmarks of Alzheimer’s disease (AD) are the aggregates of amyloid-β (Aβ) peptides and tau protein. Autophagy is a major cellular pathway leading to the removal of aggregated proteins. We have reported recently that autophagy was responsible for amyloid precursor protein cleaved C-terminal fragment (APP-CTF) degradation and amyloid β clearance in an Atg5-dependent manner. Here we aimed to elucidate the molecular mechanism by which autophagy mediates the degradation of APP-CTF and the clearance of amyloid β. Through affinity purification followed by mass spectrum analysis, we identified adaptor protein (AP) 2 together with phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) as binding proteins of microtubule-associated protein 1 light chain 3 (LC3). Further analysis showed that AP2 regulated the cellular levels of APP-CTF. Knockdown of AP2 reduced autophagy-mediated APP-CTF degradation. Immunoprecipitation and live imaging analysis demonstrated that AP2 and PICALM cross-link LC3 with APP-CTF. These data suggest that the AP-2/PICALM complex functions as an autophagic cargo receptor for the recognition and shipment of APP-CTF from the endocytic pathway to the LC3-marked autophagic degradation pathway. This molecular mechanism linking AP2/PICALM and AD is consistent with genetic evidence indicating a role for PICALM as a risk factor for AD.

Norbin is an endogenous regulator of metabotropic glutamate receptor 5 signaling.

Norbin Knockout Metabotropic glutamate receptors (mGluRs) are critical neurotransmitter sensors implicated in central neuronal functions like learning and memory and in diseases of the nervous system. Wang et al. (p. 1554) searched for proteins that interact with mGluR5a and identified a previously unrecognized component of the receptor signaling complex. The protein Norbin directly interacted with the receptor. Loss of Norbin in mice or cultured cells showed that it is necessary for the accumulation of mGluR5a in the cell membrane, for normal modulation of synaptic plasticity, and for some behavioral responses. The protein Norbin regulates the accumulation of a neurotransmitter receptor in mouse brain cell membranes. Metabotropic glutamate receptor 5 (mGluR5) is highly expressed in the mammalian central nervous system (CNS). It is involved in multiple physiological functions and is a target for treatment of various CNS disorders, including schizophrenia. We report that Norbin, a neuron-specific protein, physically interacts with mGluR5 in vivo, increases the cell surface localization of the receptor, and positively regulates mGluR5 signaling. Genetic deletion of Norbin attenuates mGluR5-dependent stable changes in synaptic function measured as long-term depression or long-term potentiation of synaptic transmission in the hippocampus. As with mGluR5 knockout mice or mice treated with mGluR5-selective antagonists, Norbin knockout mice showed a behavioral phenotype associated with a rodent model of schizophrenia, as indexed by alterations both in sensorimotor gating and psychotomimetic-induced locomotor activity.