Synapses and Dendritic Spines as Pathogenic Targets in Alzheimer's Disease

Synapses are sites of cell-cell contacts that transmit electrical or chemical signals in the brain. Dendritic spines are protrusions on dendritic shaft where excitatory synapses are located. Synapses and dendritic spines are dynamic structures whose plasticity is thought to underlie learning and memory. No wonder neurobiologists are intensively studying mechanisms governing the structural and functional plasticity of synapses and dendritic spines in an effort to understand and eventually treat neurological disorders manifesting learning and memory deficits. One of the best-studied brain disorders that prominently feature synaptic and dendritic spine pathology is Alzheimer's disease (AD). Recent studies have revealed molecular mechanisms underlying the synapse and spine pathology in AD, including a role for mislocalized tau in the postsynaptic compartment. Synaptic and dendritic spine pathology is also observed in other neurodegenerative disease. It is possible that some common pathogenic mechanisms may underlie the synaptic and dendritic spine pathology in neurodegenerative diseases

Gephyrin and the postsynaptic GABAergic complex

Although gephyrin is an important postsynaptic scaffolding protein at GABAergic synapses, the role of gephyrin for GABAergic synapse formation and/or maintenance is still under debate. In order to address this question, I manipulated the gephyrin expression levels by using small hairpin RNAs (shRNAs) to decrease gephyrin expression, by overexpressing a gephyrin-EGFP fusion protein to decrease the gephyrin clustering and by overexpression of wild type gephyrin in cultured hippocampal pyramidal cells to detect their effects on synapses. I found that decreased postsynaptic gephyrin clustering led to not only decreased postsynaptic GABA(A) receptor clusters but also decreased GABAergic innervation. It is concluded that gephyrin plays a critical role for the stability of GABAergic synapses. ^ Further more, I showed that altering the levels of expression/clustering of gephyrin also affects glutamatergic synapses. Decreasing gephyrin clusters led to increased size but not density of both the postsynaptic glutamatergic PSD-95 clusters and the presynaptic glutamatergic terminals contacting the pyramidal cells. Overexpression of gephyrin led to a slight decrease in size but not in density of both PSD-95 clusters and presynaptic glutamatergic terminals. However, the size or density of the glutamate GluR1-AMPA, or NR1-NMDA receptor clusters was not affected. It is concluded that the expression and clustering of gephyrin, a postsynaptic GABAergic scaffold protein, also plays a role in the control of the size of glutamatergic contacts. ^ I also showed that GRIP1c4-7 is a short splice form of GRIP1a, present in rat genome. The N-terminal sequence specifies its membrane localization. GRIP1c4-7 and GRIP1a/b interact with gephyrin by co-precipitation from rat brain extracts and from extracts of HEK293 cells. Moreover, purified gephyrin binds to purified GRIP1c4-7 or GRIP1a/b as shown by an affinity pull down assay, indicating that these molecules directly interact with each other. Postsynaptic gephyrin clusters colocalized with GRIP1c4-7 and GRIP1a/b clusters in cultured hippocampal neurons. These results indicate that GRIP1 proteins could be involved in the trafficking and recycling of postsynaptic gephyrin at GABAergic synapses. ^ By using gamma2-GABAAR shRNAs, an approach which is different from the knockout mice, we have shown that gamma2 subunit is important for the clustering of beta2/3-GABAARs and gephyrin. More importantly, we have shown that disrupting normal postsynaptic gamma2-GABAAR clustering leads to a significant reduction in the GABAergic innervation. The studies were done in cultured hippocampal pyramidal cells. I further extended the research to in vivo studies by transfecting neurons with in utero electroporation where I found that decreased GABA(A)R clustering and GABAergic innervation of pyramidal neurons in the post-natal rat cerebral cortex after in utero transfection of these neurons with the gamma2 shRNAs. All these results support the hypothesis that γ2 subunit is essential for the postsynaptic clustering of the GABAAR and the postsynaptic clustering of GABAAR is essential for the stability of the GABAergic synapses. ^ All these studies put together show: (I) gephyrin is critical for the postsynaptic clustering of many GABAARs; (II) γ2-GABA AR clustering is critical for the postsynaptic clustering of gephyrin and (III) the postsynaptic clustering of GABAAR and gephryin is cirtical for the stability of the presynaptic GABAergic innervation.

Gephyrin and the postsynaptic GABAergic complex

Although gephyrin is an important postsynaptic scaffolding protein at GABAergic synapses, the role of gephyrin for GABAergic synapse formation and/or maintenance is still under debate. In order to address this question, I manipulated the gephyrin expression levels by using small hairpin RNAs (shRNAs) to decrease gephyrin expression, by overexpressing a gephyrin-EGFP fusion protein to decrease the gephyrin clustering and by overexpression of wild type gephyrin in cultured hippocampal pyramidal cells to detect their effects on synapses. I found that decreased postsynaptic gephyrin clustering led to not only decreased postsynaptic GABA(A) receptor clusters but also decreased GABAergic innervation. It is concluded that gephyrin plays a critical role for the stability of GABAergic synapses. ^ Further more, I showed that altering the levels of expression/clustering of gephyrin also affects glutamatergic synapses. Decreasing gephyrin clusters led to increased size but not density of both the postsynaptic glutamatergic PSD-95 clusters and the presynaptic glutamatergic terminals contacting the pyramidal cells. Overexpression of gephyrin led to a slight decrease in size but not in density of both PSD-95 clusters and presynaptic glutamatergic terminals. However, the size or density of the glutamate GluR1-AMPA, or NR1-NMDA receptor clusters was not affected. It is concluded that the expression and clustering of gephyrin, a postsynaptic GABAergic scaffold protein, also plays a role in the control of the size of glutamatergic contacts. ^ I also showed that GRIP1c4-7 is a short splice form of GRIP1a, present in rat genome. The N-terminal sequence specifies its membrane localization. GRIP1c4-7 and GRIP1a/b interact with gephyrin by co-precipitation from rat brain extracts and from extracts of HEK293 cells. Moreover, purified gephyrin binds to purified GRIP1c4-7 or GRIP1a/b as shown by an affinity pull down assay, indicating that these molecules directly interact with each other. Postsynaptic gephyrin clusters colocalized with GRIP1c4-7 and GRIP1a/b clusters in cultured hippocampal neurons. These results indicate that GRIP1 proteins could be involved in the trafficking and recycling of postsynaptic gephyrin at GABAergic synapses. ^ By using gamma2-GABAAR shRNAs, an approach which is different from the knockout mice, we have shown that gamma2 subunit is important for the clustering of beta2/3-GABAARs and gephyrin. More importantly, we have shown that disrupting normal postsynaptic gamma2-GABAAR clustering leads to a significant reduction in the GABAergic innervation. The studies were done in cultured hippocampal pyramidal cells. I further extended the research to in vivo studies by transfecting neurons with in utero electroporation where I found that decreased GABA(A)R clustering and GABAergic innervation of pyramidal neurons in the post-natal rat cerebral cortex after in utero transfection of these neurons with the gamma2 shRNAs. All these results support the hypothesis that γ2 subunit is essential for the postsynaptic clustering of the GABAAR and the postsynaptic clustering of GABAAR is essential for the stability of the GABAergic synapses. ^ All these studies put together show: (I) gephyrin is critical for the postsynaptic clustering of many GABAARs; (II) γ2-GABA AR clustering is critical for the postsynaptic clustering of gephyrin and (III) the postsynaptic clustering of GABAAR and gephryin is cirtical for the stability of the presynaptic GABAergic innervation.

The Plant Nucleoskeleton: Ultrastructural Organization and Identification of NuMA Homologues in the Nuclear Matrix and Mitotic Spindle of Plant Cells

In the present work we investigate the structural organization of the nucleoskeleton ofAllium cepameristematic root cells. Resinless sections reveal for the first time a residual filamentous network in plant nuclei. This network is composed of branched knobbed filaments with associated globular structures, connected to the lamina and to the dense aggregates of different sizes. Results of immunoblotting show that many components of this network are homologues of intermediate filament-type proteins. NuMA, a coiled-coil protein related to intermediate filaments, found in animal cells, can also be detected in this plant nuclear matrix system. Immunofluorescence reveals a diffuse distribution of the animal NuMA homologues in plant nuclear core filaments in interphase. Resinless immunoelectron microscopy further reveals a distribution along the extended filaments and the dense aggregates. During mitosis, in contrast to the accumulation at the poles in animal cells, NuMA homologues in plant onion cells show a diffuse pattern, which may correspond to the spindle matrix. Our data are the first report of the conservation in plants of NuMA proteins, which may be involved in both nuclear and mitotic spindle organizations.This work has been supported by the Spanish DGES (ProjectsPB95-0117 and PB96-0909). The Spanish Agency of Co-operation(AECI) through the Spanish-Chinese Intergovernmental Agreementhas financed Dr. W. Yu.Peer reviewe

The PINK1/Parkin pathway regulates mitochondrial dynamics and function in mammalian hippocampal and dopaminergic neurons

PTEN-induced putative kinase 1 (PINK1) and Parkin act in a common pathway to regulate mitochondrial dynamics, the involvement of which in the pathogenesis of Parkinson's disease (PD) is increasingly being appreciated. However, how the PINK1/Parkin pathway influences mitochondrial function is not well understood, and the exact role of this pathway in controlling mitochondrial dynamics remains controversial. Here we used mammalian primary neurons to examine the function of the PINK1/Parkin pathway in regulating mitochondrial dynamics and function. In rat hippocampal neurons, PINK1 or Parkin overexpression resulted in increased mitochondrial number, smaller mitochondrial size and reduced mitochondrial occupancy of neuronal processes, suggesting that the balance of mitochondrial fission/fusion dynamics is tipped toward more fission. Conversely, inactivation of PINK1 resulted in elongated mitochondria, indicating that the balance of mitochondrial fission/fusion dynamics is tipped toward more fusion. Furthermore, overexpression of the fission protein Drp1 (dynamin-related protein 1) or knocking down of the fusion protein OPA1 (optical atrophy 1) suppressed PINK1 RNAi-induced mitochondrial morphological defect, and overexpression of PINK1 or Parkin suppressed the elongated mitochondria phenotype caused by Drp1 RNAi. Functionally, PINK1 knockdown and overexpression had opposite effects on dendritic spine formation and neuronal vulnerability to excitotoxicity. Finally, we found that PINK1/Parkin similarly influenced mitochondrial dynamics in rat midbrain dopaminergic neurons. These results, together with previous findings in Drosophila dopaminergic neurons, indicate that the PINK1/Parkin pathway plays conserved roles in regulating neuronal mitochondrial dynamics and function

Phospho-dependent ubiquitination and degradation of PAR-1 regulates synaptic morphology and tau-mediated Aβ toxicity in Drosophila

The conserved kinases PAR-1/MARK are critically involved in processes such as asymmetric cell division, cell polarity and neuronal differentiation. Their deregulation has been implicated in diseases including Alzheimer's disease and cancer. Given the importance of PAR-1/MARK in health and disease, their activities need to be tightly controlled. However, little is known about the molecular mechanisms underlying their regulation in vivo. Here we show that in Drosophila, a phosphorylation-dependent ubiquitination mechanism restrains PAR-1 activation. Active PAR-1 generated by LKB1-controlled phosphorylation is targeted for ubiquitination and degradation by SCF (Skp, Cullin, F-box containing complex) (Slimb), whose action is antagonized by the deubiquitinating enzyme fat facets. This newly identified PAR-1-modifying module critically regulates synaptic morphology and tau-mediated postsynaptic toxicity of amyloid precursor protein (APP)/Aβ-42, the causative agents of Alzheimer's disease, at the Drosophila neuromuscular junction. Our results provide new insights into the regulation of PAR-1 in various physiological processes and offer new therapeutic strategies for diseases involving PAR-1/MARK deregulation

Spontaneous and light-induced photon emission from intact brains of chick embryos

Photon emission (PE) and light-induced photon emission(LPE) of intact brains isolated from chick embryos have been measured by using the single photon counting device. Experimental results showed that the intensity level of photon emission was detected to be higher from intact brain than from the medium in which the brain was immerged during measuring, and the emission intensity was related to the developmental stages, the healthy situation of the measured embryos, and the freshness of isolated brains as well. After white light illumination, a short-life delayed emission from intact brains was observed, and its relaxation behavior followed a hyperbolic rather than an exponential law. According to the hypothesis of biophoton emission originating from a delocalized coherent electromagnetic field and Frohlich’ s idea of coherent long-range interactions in biological systems, discussions were made on the significance of photon emission in studying cell communication, biological regulation, living system’s relevance to its environment, and also on the relations between the detected photon emission and the coherent electromagnetic field. The detected photon emission should be comprehended in the manner of the interactions between the intrinsic fields within the living systems and their environmental external fields

A critical role for the PAR-1/MARK-tau axis in mediating the toxic effects of Aβ on synapses and dendritic spines

Alzheimer's disease (AD) is the most common neurodegenerative disease and the leading cause of dementia in the elderly. Accumulating evidence supports soluble amyloid-β (Aβ) oligomers as the leading candidate for the causative agent in AD and synapses as the primary site of Aβ oligomer action. However, the molecular and cellular mechanisms by which Aβ oligomers cause synaptic dysfunction and cognitive impairments remain poorly understood. Using primary cultures of rat hippocampal neurons as a model system, we show that the partitioning defective-1 (PAR-1)/microtubule affinity-regulating kinase (MARK) family kinases act as critical mediators of Aβ toxicity on synapses and dendritic spines. Overexpression of MARK4 led to tau hyperphosphorylation, reduced expression of synaptic markers, and loss of dendritic spines and synapses, phenotypes also observed after Aβ treatment. Importantly, expression of a non-phosphorylatable form of tau with the PAR-1/MARK site mutated blocked the synaptic toxicity induced by MARK4 overexpression or Aβ treatment. To probe the involvement of endogenous MARK kinases in mediating the synaptic toxicity of Aβ, we employed a peptide inhibitor capable of effectively and specifically inhibiting the activities of all PAR-1/MARK family members. This inhibitor abrogated the toxic effects of Aβ oligomers on dendritic spines and synapses as assayed at the morphological and electrophysiological levels. Our results reveal a critical role for PAR-1/MARK kinases in AD pathogenesis and suggest PAR-1/MARK inhibitors as potential therapeutics for AD and possibly other tauopathies where aberrant tau hyperphosphorylation is involved