Identification of novel Ack1-interacting proteins and Ack1 phosphorylated sites in mouse brain by mass spectrometry.

Ack1 (activated Cdc42-associated tyrosine kinase) is a non-receptor tyrosine kinase that is highly expressed in brain. This kinase contains several protein-protein interaction domains and its action is partially regulated by phosphorylation. As a first step to address the neuronal functions of Ack1, here we screened mouse brain samples to identify proteins that interact with this kinase. Using mass spectrometry analysis, we identified new putative partners for Ack1 including cytoskeletal proteins such as Drebrin or MAP4; adhesion regulators such as NCAM1 and neurabin-2; and synapse mediators such as SynGAP, GRIN1 and GRIN3. In addition, we confirmed that Ack1 and CAMKII both co-immunoprecipitate and co-localize in neurons. We also identified that adult and P5 samples contained the phosphorylated residues Thr 104 and Ser 825, and only P5 samples contained phosphorylated Ser 722, a site linked to cancer and interleukin signaling when phosphorylated. All these findings support the notion that Ack1 could be involved in neuronal plasticity.

New partners and phosphorylation sites of focal adhesion kinase identified by mass spectrometry.

The regulation of focal adhesion kinase (FAK) involves phosphorylation and multiple interactions with other signaling proteins. Some of these pathways are relevant for nervous system functions such as branching, axonal guidance, and plasticity. In this study, we screened mouse brain to identify FAK-interactive proteins and phosphorylatable residues as a first step to address the neuronal functions of this kinase. Using mass spectrometry analysis, we identified new phosphorylated sites (Thr 952, Thr 1048, and Ser 1049), which lie in the FAT domain; and putative new partners for FAK, which include cytoskeletal proteins such as drebrin and MAP 6, adhesion regulators such as neurabin-2 and plakophilin 1, and synapse-associated proteins such as SynGAP and a NMDA receptor subunit. Our findings support the participation of brain-localized FAK in neuronal plasticity.

Bcl-2 overexpression delays caspase-3 activation and rescues cerebellar degeneration in prion-deficient mice that overexpress amino-terminally truncated prion.

Prnp knockout mice that overexpress an amino-truncated form of PrPc (deltaPrP) are ataxic and display cerebellar cell loss and premature death. Studies on the molecular and intracellular events that trigger cell death in these mutants may contribute to elucidate the functions of PrPc and to the design of treatments for prion disease. Here we examined the effects of Bcl-2 overexpression in neurons on the development of the neurological syndrome and cerebellar pathology of deltaPrP. We show that deltaPrP overexpression activates the stress-associated kinases ERK1-2 in reactive astroglia, p38 and the phosphorylation of p53, which leads to the death of cerebellar neurons in mutant mice. We found that the expression of deltaPrP in cell lines expressing very low levels of PrPc strongly induces the activation of apoptotic pathways, thereby leading to caspase-3 activation and cell death, which can be prevented by coexpressing Bcl-2. Finally, we corroborate in vivo that neuronal-directed Bcl-2 overexpression in deltaPrP mice (deltaPrP Bcl-2) markedly reduces caspase-3 activation, glial activation, and neuronal cell death in cerebellum by improving locomotor deficits and life expectancy.

Nogo-A expression in the human hippocampus in normal aging and in Alzheimer disease.

Myelin-associated proteins are involved in the formation and stabilization of myelin sheaths. In addition, they prevent axon regeneration and plasticity in the adult brain. Recent evidence suggests that the expression of certain myelin-associated proteins (e.g. Nogo-A) can be regulated by synaptic activity or by over-expression after neural lesions in brain syndromes such as temporal lobe epilepsy. However, no studies on Alzheimer disease (AD) have been reported in which cell loss and significant synaptic reorganization occurs. In the present study, we analyze in detail the expression of Nogo-A in the hippocampal formation in normal human aging and in AD. Our results indicate that Nogo-A is expressed by oligodendrocytes and neurons in the aged hippocampal formation. In addition, both granule cells and mossy fiber connections are also labeled in the old-aged hippocampi. Interestingly, Nogo-A is over-expressed by hippocampal neurons in AD and is associated with beta-amyloid deposits in senile plaques. Taken together, our results reinforce the hypothesis that Reticulon proteins such as Nogo-A participate in the neuronal responses stemming from hippocampal formation during senescence, and particularly in AD. These findings also indicate that Reticulon proteins could be considered as new putative drug targets in therapies of neurodegenerative disorders.

Expression pattern of ACK1 tyrosine kinase during brain development in the mouse.

Ack1 is a non-receptor tyrosine kinase that is highly expressed in the adult central nervous system (CNS). Here, we studied the distribution of Ack1 mRNA throughout the development of mouse CNS. Expression was detected in all areas of the brain but especially high levels were observed in the neocortex, hippocampus, and cerebellum. Interestingly, expression levels were prominent in areas of proliferation such as the subventricular zone and areas that originate other structures such the pontine nucleus and the ganglionic eminence. During development, several areas showed an increase in Ack1 expression, especially the dentate gyrus and CA3 in the hippocampus, layer V in the neocortex, and the Purkinje cell layer in the cerebellum. These results demonstrate that this kinase is up-regulated during development and that it is expressed in proliferative areas and in migratory pathways in the developing brain.

Expression, synaptic localization, and developmental regulation of Ack1/Pyk1, a cytoplasmic tyrosine kinase highly expressed in the developing and adult brain.

Cytosolic tyrosine kinases play a critical role both in neural development and in adult brain function and plasticity. Here we isolated a cDNA with high homology to human Ack1 and mouse Tnk2. This cDNA directs the expression of a 125-kD protein that can be autophosphorylated in tyrosines. Initially, this clone was named Pyk1 for proline-rich tyrosine kinase (Lev et al., 1995); however, since it corresponds to the mouse homolog of Ack1, here we called it Ack1/Pyk1. In this study we show that Ack1/Pyk1 mRNA and protein is highly expressed in the developing and adult brain. The highest levels of Ack1/Pyk1 expression were detected in the hippocampus, neocortex, and cerebellum. Electron microscopy studies showed that Ack1/Pyk1 protein is expressed in these regions both at dendritic spines and presynaptic axon terminals, indicating a role in synaptic function. Furthermore, we demonstrate that Ack1/Pyk1 mRNA levels are strongly upregulated by increased neural activity, produced by intraperitoneal kainate injections. During development, Ack1/Pyk1 was also expressed in the proliferative ventricular zones and in postmitotic maturing neurons. In neuronal cultures, Ack1/Pyk1 was detected in developing dendrites and axons, including dendritic tips and growth cones. Moreover, Ack1/Pyk1 colocalized with Cdc42 GTPase in neuronal cultures and coimmunoprecipitated with Cdc42 in HEK 293T cells. Altogether, our findings indicate that Ack1/Pyk1 tyrosine kinase may be involved both in adult synaptic function and plasticity and in brain development.

PrP(106-126) activates neuronal intracellular kinases and Egr1 synthesis through activation of NADPH-oxidase independently of PrPc.

Prion diseases are characterised by severe neural lesions linked to the presence of an abnormal protease-resistant isoform of cellular prion protein (PrPc). The peptide PrP(106-126) is widely used as a model of neurotoxicity in prion diseases. Here, we examine in detail the intracellular signalling cascades induced by PrP(106-126) in cortical neurons and the participation of PrPc. We show that PrP(106-126) induces the activation of subsets of intracellular kinases (e.g., ERK1/2), early growth response 1 synthesis and induces caspase-3 activity, all of which are mediated by nicotinamide adenine dinucleotide phosphate hydrogen-oxidase activity and oxidative stress. However, cells lacking PrPc are similarly affected after peptide exposure, and this questions the involvement of PrPc in these effects.