Xin-Ran Zhu

Frequenin—A novel calcium-binding protein that modulates synaptic efficacy in the drosophila nervous system

The T(X;Y)V7 rearrangement in Drosophila has originally been recognized as a Shaker-like mutant because of its behavioral and electrophysiological phenotype. The gene whose expression is altered by the V7 rearrangement has been characterized. It encodes a novel Ca(2+)-binding protein named frequenin, which is related to recoverin and visinin. In vitro, the frequenin protein functions like recoverin as a Ca(2+)-sensitive guanylyl cyclase activator. Anti-frequenin antibodies stain the central and peripheral nervous system in Drosophila embryos and in larval and adult tissue sections. Frequenin appears to be particularly enriched in synapses, such as the motor nerve endings at neuromuscular junctions. Neuromuscular junctions of transgenic flies, which overexpress frequenin upon heat shock, exhibit an extraordinarily enhanced, frequency-dependent facilitation of neurotransmitter release, with properties identical to those observed in V7 junctions. We propose that frequenin represents a new element for the Ca(2+)-dependent modulation of synaptic efficacy.

Integrin-mediated short-term memory in Drosophila

Volado is a new memory mutant of Drosophila. The locus encodes two isoforms of a new α-integrin, a molecule that dynamically mediates cell adhesion and signal transduction. The Volado gene is expressed preferentially in mushroom body cells, which are neurons known to mediate olfactory learning in insects. Volado proteins are concentrated in the mushroom body neuropil, brain areas that contain mushroom body processes in synaptic contact with other neurons. Volado mutants display impaired olfactory memories within 3 min of training, indicating that the integrin is required for short-term memory processes. Conditional expression of a Volado transgene during adulthood rescues the memory impairment. This rescue of memory is reversible, fading over time along with expression of the transgene. Thus the Volado integrin is essential for the physiological processes underlying memory. We propose a model in which integrins act as dynamic regulators of synapse structure or the signalling events underlying short-term memory formation.

Parkin expression in the adult mouse brain.

Mutations in a protein designated Parkin were shown to be involved in the pathogenesis of autosomal recessive juvenile parkinsonism. Nothing is known about its regional and subcellular distribution in the mouse. In order to elucidate the Parkin mRNA and protein distribution in the adult mouse, the mouse cDNA was cloned and polyclonal antisera were generated against the N‐terminal part of mouse Parkin. The antibodies were shown to be specific using Western blot analysis, immunostaining of cells transfected with mouse Parkin and pre‐absorption tests. The Parkin protein expression profile was studied using immunohistochemistry and Western blot analysis and was compared with that of the mRNA yielded by in situ hybridization and RT‐PCR analysis. Parkin protein was widely distributed in all subdivisions of the mouse brain. Low levels were found in the telencephalon and diencephalon, while the brainstem contained a large number of cells heavily expressing Parkin. Ultrastructural analysis and double immunohistochemistry revealed that the majority of Parkin‐expressing cells were neurons, while only single glial cells exhibited immunostaining. The protein was distributed nonhomogeneously throughout the entire cytoplasm. A subpopulation of Parkin‐immunopositive cells displayed speckled immunodeposits in the nucleus. Dopaminergic cells of the substantia nigra pars compacta exhibited high levels of Parkin mRNA but no Parkin protein, while the striatum contained immunopositive profiles but no mRNA signals. Our data indicate that Parkin is neither restricted to a single functional system nor associated with a particular transmitter system. The speckled nuclear distribution of Parkin immunoreactivity strongly suggests a role for Parkin in gene expression.

Non-motor behavioural impairments in parkin-deficient mice.

Mutations in the parkin gene are the major cause of early‐onset familial Parkinsons disease (PD). We previously reported the generation and analysis of a knockout mouse carrying a deletion of exon 3 in the parkin gene. F1 hybrid pa+/– mice were backcrossed to wild‐type C57Bl/6 for three more generations to establish a pa–/–(F4) mouse line. The appearance of tyrosine hydroxylase‐positive neurons was normal in young and aged pa–/– (F4) animals. Loss of parkin function in mice did not enhance vulnerability of dopaminergic neurons to 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) toxicity. However, the pa–/– (F4) mice displayed impaired exploration and habituation to a new environment and exhibited thigmotaxis behaviour in the open field and Morris water maze. Abnormal anxiety‐related behaviour of pa–/– (F4) mice was also observed in the light/dark exploration test paradigm. Dopamine metabolism was enhanced in the striatum of pa–/– (F4) mice, as revealed by increased homovanillic acid (HVA) content and a reduced ratio of dihydroxyphenylacetic acid (DOPAC)/HVA. The alterations found in the dopaminergic system could be responsible for the behavioural impairments of pa–/– (F4) mice. Consistent with a recent observation of cognitive dysfunction in parkin‐linked patients with PD, our findings provide evidence of a physiological role of parkin in non‐motor behaviour, possibly representing a disease stage that precedes dopaminergic neuron loss.

Expression of DJ-1 in the adult mouse CNS

Recessively inherited mutations in DJ-1 have recently been linked to familial forms of parkinsonism. However, the regional and cellular expression of DJ-1 is largely unknown. In the present study, we mapped the distribution of DJ-1 transcript and protein by non-radioactive in situ hybridization and immunohistochemistry in adult mouse CNS. For immunohistochemical analysis, we raised a polyclonal antiserum against GST-mouse DJ-1 fusion protein. DJ-1 transcript and protein were strongly and homogenously expressed in all CNS regions. Colocalization experiments revealed expression in neurons of different neurotransmitter phenotypes and in all glial cell types, such as astrocytes, microglia and oligodendrocytes. DJ-1 protein was diffusely distributed throughout the cytoplasma of the soma and the proximal parts of the processes, while the nuclei were always spared. The high expression of DJ-1 in neuronal and glial cells, that is not confined to a single functional system or any anatomical area, supports the view of a basic physiological role in cell biology.

Sgk1, a cell survival response in neurodegenerative diseases.

Serum and glucocorticoid-regulated kinase 1 (sgk1) belongs to a family of serine/threonine kinases that is under acute transcriptional control by serum and glucocorticoids. An expanding set of receptors and cellular stress pathways has been shown to enhance sgk1 expression, which is implicated in the regulation of ion channel conductance, cell volume, cell cycle progression, and apoptosis. Recent evidence for the involvement of sgk1 in the early pathogenesis of MPTP-induced Parkinsons disease (PD) prompted us to investigate in more detail its expression and role in animal models of different neurodegenerative diseases. Here, we show that transcription of sgk1 is increased in several animal models of PD and a transgenic model of amyotrophic lateral sclerosis (ALS). The upregulation of sgk1 strongly correlates with the occurrence of cell death. Furthermore, we provide evidence that the Forkhead transcription factor FKHRL1 and some of the voltage-gated potassium channels are physiological substrates of sgk1 in vivo. Using a small interfering RNA approach to silence sgk1 transcripts in vitro, we give evidence that sgk1 exerts a protective role in oxidative stress situations. These findings underline a key role for sgk1 in the molecular pathway of cell death, in which sgk1 seems to exert a protective role.

Fragrant dioxane derivatives identify β1 subunit-containing GABAA receptors.

Nineteen GABAA receptor (GABAAR) subunits are known in mammals with only a restricted number of functionally identified native combinations. The physiological role of β1-subunit-containing GABAARs is unknown. Here we report the discovery of a new structural class of GABAAR positive modulators with unique β1-subunit selectivity: fragrant dioxane derivatives (FDD). At heterologously expressed α1βxγ2L (x-for 1,2,3) GABAAR FDD were 6 times more potent at β1- versus β2- and β3-containing receptors. Serine at position 265 was essential for the high sensitivity of the β1-subunit to FDD and the β1N286W mutation nearly abolished modulation; vice versa the mutation β3N265S shifted FDD sensitivity toward the β1-type. In posterior hypothalamic neurons controlling wakefulness GABA-mediated whole-cell responses and GABAergic synaptic currents were highly sensitive to FDD, in contrast to β1-negative cerebellar Purkinje neurons. Immunostaining for the β1-subunit and the potency of FDD to modulate GABA responses in cultured hypothalamic neurons was drastically diminished by β1-siRNA treatment. In conclusion, with the help of FDDs we reveal a functional expression of β1-containing GABAARs in the hypothalamus, offering a new tool for studies on the functional diversity of native GABAARs.

Differential expression of cathepsin X in aging and pathological central nervous system of mice

Increasing evidence of a fundamental influence of cathepsins on inflammation has drawn interest in a thorough understanding of their role in physiological and pathological processes. Even though the number of identified cathepsins has more than doubled in the last years, information about their expression, regulation and function in the brain is still incomplete. In the present study we analyzed the regional, cellular and subcellular localization and the activity of the recently discovered cathepsin X in the normal, developing and pathological mouse brain. Our results show that CATX is: (i) is expressed in almost all cells in the mouse brain with a preference for glial cells; (ii) already widely expressed early in development and age-dependently upregulated in amount and activity; (iii) prominently localized in the lysosomal system but also scattered in the somal cytoplasm in the aged brain; (iv) upregulated in numerous glial cells of degenerating brain regions in a transgenic mouse model of amyotrophic lateral sclerosis; and (v) associated with plaques in a transgenic mouse model and in Alzheimer patients. These results strongly suggest that cathepsin X is an important player in degenerative processes during normal aging and in pathological conditions.

The neuronal RhoA GEF, Tech, interacts with the synaptic multi-PDZ-domain-containing protein, MUPP1

Tech is a RhoA guanine nucleotide exchange factor (GEF) that is highly enriched in hippocampal and cortical neurons. To help define its function, we have conducted studies aimed at identifying partner proteins that bind to its C‐terminal PDZ ligand motif. Yeast two hybrid studies using the Tech C‐terminal segment as bait identified MUPP1, a protein that contains 13 PDZ domains and has been localized to the post‐synaptic compartment, as a candidate partner protein for Tech. Co‐transfection of Tech and MUPP1 in human embryonic kidney 293 cells confirmed that these full‐length proteins interact in a PDZ‐dependent fashion. Furthermore, we confirmed that endogenous Tech co‐precipitates with MUPP1, but not PSD‐95, from hippocampal and cortical extracts prepared from rat brain. In addition, immunostaining of primary cortical cultures revealed co‐localization of MUPP1 and Tech puncta in the vicinity of synapses. In assessing which PDZ domains of MUPP1 mediate binding to Tech, we found that Tech can bind to either PDZ domain 10 or 13 of MUPP1 as mutation of both these domains is needed to disrupt their interaction. Taken together, these findings demonstrate that Tech binds to MUPP1 and suggest that it regulates RhoA signaling pathways in the vicinity of synapses.

Influence of Different Promoters on the Expression Pattern of Mutated Human α-Synuclein in Transgenic Mice

Two missense mutations (A53T and A30P) in the gene encoding the presynaptic protein α-synuclein (asyn) are associated with rare, dominantly inherited forms of Parkinson’s disease (PD) and its accumulation in Lewy bodies and Lewy neurites. As an initial step in investigating the role of asyn in the pathogenesis of PD, we have generated C57BL/6 transgenic mice overexpressing the doubly mutated human asyn under the control of three different promoters; the chicken β-actin (chβactin), the mouse tyrosine hydroxylase 9.6 kb (msTH) and the mouse prion protein (msprp). In this study we compared the regional and cellular expression pattern of the transgenic protein in the brain and peripheral organs of various transgenic mouse lines. Western blot analysis and immunohistochemistry consistently showed that all three promoters successfully drive the expression of the transgene. The msprp promoter was found to give the highest level of transgene expression. All promoters directed the expression into the brain and specific neuron types. However, the promoters differed with respect to (i) the expression pattern in peripheral organs, (ii) the number and (iii) the regional distribution of expressing cells in the brain. Furthermore, remarkable line-to-line variation of expression patterns was observed in mouse lines carrying the same construct. Future studies will analyze how the variations in transgene expression affect the pathogenesis in the animals.