Phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) and PARK2/Parkin mutations cause autosomal recessive forms of Parkinson's disease. Upon a loss of mitochondrial membrane potential (ΔΨ m ) in human cells, cytosolic Parkin has been reported to be recruited to mitochondria, which is followed by a stimulation of mitochondrial autophagy. Here, we show that the relocation of Parkin to mitochondria induced by a collapse of ΔΨ m relies on PINK1 expression and that overexpression of WT but not of mutated PINK1 causes Parkin translocation to mitochondria, even in cells with normal ΔΨ m . We also show that once at the mitochondria, Parkin is in close proximity to PINK1, but we find no evidence that Parkin catalyzes PINK1 ubiquitination or that PINK1 phosphorylates Parkin. However, co-overexpression of Parkin and PINK1 collapses the normal tubular mitochondrial network into mitochondrial aggregates and/or large perinuclear clusters, many of which are surrounded by autophagic vacuoles. Our results suggest that Parkin, together with PINK1, modulates mitochondrial trafficking, especially to the perinuclear region, a subcellular area associated with autophagy. Thus by impairing this process, mutations in either Parkin or PINK1 may alter mitochondrial turnover which, in turn, may cause the accumulation of defective mitochondria and, ultimately, neurodegeneration in Parkinson's disease.autophagy | Parkinson's disease | phosphatase and tensin homolog-induced putative kinase 1 T he common neurodegenerative disorder Parkinson's disease (PD) occasionally can be inherited (1, 2). Parkinson disease 6/ phosphatase and tensin homolog (PTEN)-induced putative kinase-1 (PARK6/PINK1) is among the gene products associated with familial PD (2, 3). This 581-amino acid polypeptide is localized to the mitochondria and has only a single recognized functional domain, a serine/threonine kinase with a high degree of homology to that of the Ca 2+ /calmodulin kinase family. Overexpression of WT PINK1 rescues abnormal mitochondrial morphology that has been described in Drosophila carrying Pink1 mutations (4, 5), a finding that supports the notion that the mutated allele gives rise to a loss-of-function phenotype. Loss-offunction mutations in the gene encoding PARK2/Parkin (an E3 ubiquitin ligase) also can cause an autosomal recessive form of familial PD (2, 6). Parkin is thought to operate within the same molecular pathway as PINK1 to modulate mitochondrial dynamics (4, 5, 7). This possibility is intriguing, because Parkin has been reported to be essentially cytosolic (8, 9). However, we have shown that PINK1 spans the outer mitochondrial membrane, with its kinase domain facing the cytoplasm (10). These details of PINK1 topology are relevant to the reported Parkin/PINK1 genetic interaction because they place the only known functional domain of PINK1 in the same subcellular compartment as Parkin.However, the role played by Parkin, PINK1, or both in mitochondrial dynamics is still uncertain. Perhaps, the beginning of an answer to th...

Mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson’s disease. We created a LRRK2 transgenic mouse model that recapitulates cardinal features of the disease: an age-dependent and levodopa-responsive slowness of movement associated with diminished dopamine release and axonal pathology of nigrostriatal dopaminergic projection. These mice provide a valid model of Parkinson’s disease and are a resource for the investigation of pathogenesis and therapeutics.

The innate immune system relies on evolutionally conserved Toll-like receptors (TLRs) to recognize diverse microbial molecular structures. Most TLRs depend on a family of adaptor proteins termed MyD88s to transduce their signals. Critical roles of MyD88-1–4 in host defense were demonstrated by defective immune responses in knockout mice. In contrast, the sites of expression and functions of vertebrate MyD88-5 have remained elusive. We show that MyD88-5 is distinct from other MyD88s in that MyD88-5 is preferentially expressed in neurons, colocalizes in part with mitochondria and JNK3, and regulates neuronal death. We prepared MyD88-5/GFP transgenic mice via a bacterial artificial chromosome to preserve its endogenous expression pattern. MyD88-5/GFP was detected chiefly in the brain, where it associated with punctate structures within neurons and copurified in part with mitochondria. In vitro, MyD88-5 coimmunoprecipitated with JNK3 and recruited JNK3 from cytosol to mitochondria. Hippocampal neurons from MyD88-5–deficient mice were protected from death after deprivation of oxygen and glucose. In contrast, MyD88-5–null macrophages behaved like wild-type cells in their response to microbial products. Thus, MyD88-5 appears unique among MyD88s in functioning to mediate stress-induced neuronal toxicity.

Estrogen (E) treatment induces axospinous synapses in rat hippocampus in vivo and in cultured hippocampal neurons in vitro. To better explore the molecular mechanisms underlying this phenomenon, we have established a mouse model for E action in the hippocampus by using Golgi impregnation to examine hippocampal dendritic spine morphology, radioimmunocytochemistry (RICC) and silver-enhanced immunocytochemistry to examine expression levels of synaptic protein markers, and hippocampal-dependent object-placement memory as a behavioral readout for the actions of E. In ovariectomized mice of several strains and F 1 hybrids, the total dendritic spine density on neurons in the CA1 region was not enhanced by E treatment, a finding that differs from that in the female rat. E treatment of ovariectomized C57BL͞6J mice, however, caused an increase in the number of spines with mushroom shapes. By RICC and silver-enhanced immunocytochemistry, we found that the immunoreactivity of postsynaptic markers (PSD95 and spinophilin) and a presynaptic marker (syntaxin) were enhanced by E treatment throughout all fields of the dorsal hippocampus. In the object-placement tests, E treatment enhanced performance of object placement, a spatial episodic memory task. Taken together, the morphology and RICC results suggest a previously uncharacterized role of E in synaptic structural plasticity that may be interpreted as a facilitation of the spine-maturation process and may be associated with enhancement of hippocampal-dependent memory.D endritic spines are specialized to receive synaptic inputs and to compartmentalize calcium, and changes in spine morphology and function are considered to be important for processes such as learning and memory (1-5). It is, therefore, important to understand how dendritic spine formation and maturation are regulated. Extrinsic factors, such as circulating hormones, influence spine properties in the hippocampus. Estrogen (E) treatment regulates dendritic spine formation in the rat hippocampus in vivo (6-8) and in cultured hippocampal neurons in vitro (9-12). The effects of E on hippocampaldependent cognitive functions were shown also in rats and humans (13-15) and recently in mice and nonhuman primates (16)(17)(18)(19).Dendritic-spine changes include at least two different processes: generation of new spines and maturation of existing spine synapses. These processes are closely linked, with complex biochemical, morphological, and electrophysiological consequences (1,2,20). Spine maturation is a multistep, multifaceted process in which the spines change from thin filopodia-like structures to spines with bigger heads, larger synaptic contact area, shorter and wider spine necks, and newly recruited synaptic proteins (1,3,(20)(21)(22). In cell culture, only the mature type of dendritic spines can recruit ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors (2) and, thus, make the transition from silent to functional synapses (23).Studies of E-induced synapse formation in the rat hippocampus have use...

Expanded polyglutamine (polyQ) proteins in Huntington's disease (HD) as well as other polyQ disorders are known to elicit a variety of intracellular toxicities, but it remains unclear whether polyQ proteins can elicit pathological cell-cell interactions which are critical to disease pathogenesis. To test this possibility, we have created conditional HD mice expressing a neuropathogenic form of mutant huntingtin (mhtt-exon1) in discrete neuronal populations. We show that mhtt aggregation is a cell-autonomous process. However, progressive motor deficits and cortical neuropathology are only observed when mhtt expression is in multiple neuronal types, including cortical interneurons, but not when mhtt expression is restricted to cortical pyramidal neurons. We further demonstrate an early deficit in cortical inhibition, suggesting that pathological interactions between interneurons and pyramidal neurons may contribute to the cortical manifestation of HD. Our study provides genetic evidence that pathological cell-cell interactions elicited by neuropathogenic forms of mhtt can critically contribute to cortical pathogenesis in a HD mouse model.

Estrogens (E) and progestins regulate synaptogenesis in the CA1 region of the dorsal hippocampus during the estrous cycle of the female rat, and the functional consequences include changes in neurotransmission and memory. Synapse formation has been demonstrated by using the Golgi technique, dye filling of cells, electron microscopy, and radioimmunocytochemistry. N-methyl-D-aspartate (NMDA) receptor activation is required, and inhibitory interneurons play a pivotal role as they express nuclear estrogen receptor alpha (ER␣) and show E-induced decreases of GABAergic activity. Although global decreases in inhibitory tone may be important, a more local role for E in CA1 neurons seems likely. The rat hippocampus expresses both ER␣ and ER␤ mRNA. At the light microscopic level, autoradiography shows cell nuclear [ 3 H]estrogen and [ 125 I]estrogen uptake according to a distribution that primarily reflects the localization of ER␣-immunoreactive interneurons in the hippocampus. However, recent ultrastructural studies have revealed extranuclear ER␣ immunoreactivity (IR) within select dendritic spines on hippocampal principal cells, axon terminals, and glial processes, localizations that would not be detectable by using standard light microscopic methods. Based on recent studies showing that both types of ER are expressed in a form that activates second messenger systems, these findings support a testable model in which local, non-genomic regulation by estrogen participates along with genomic actions of estrogens in the regulation of synapse formation. T he brain is widely responsive to gonadal hormones. Not only is the hypothalamus regulated by these hormones in relation to reproductive behavior and neuroendocrine physiology, but also structures like the hippocampus and midbrain serotonin system undergo sexual differentiation during perinatal development and are hormone responsive in maturity (1, 2). One of the processes regulated by ovarian hormones is the cyclic formation and breakdown of excitatory synapses on dendritic spines in the hippocampus (3). This finding was surprising because, until recently, the hippocampus was known as a brain region in which cell nuclear estrogen receptors (ER) are present in scattered inhibitory interneurons but not in principal neurons where spine formation occurs (4). Yet the effects of ovarian hormones on synaptic turnover were as impressive in the hippocampus as those in the ventromedial hypothalamus (5-7), a classic estrogen (E) target area of the brain for female sexual behavior (8). Moreover, effects of estrogens on hippocampal-dependent cognitive function are now recognized in rodents (9) and humans (10).Recent electron microscopic studies have revealed that ERs are expressed in hippocampus in non-nuclear locations within principal cells (11). This fact, along with the discovery that ER can couple to second messenger systems (12-14), has raised the possibility that ER may be involved in local signaling within neurons as well as regulating expression of genes via nuclear receptors i...

Bacterial artificial chromosome (BAC) mediated transgenesis has proven to be a highly reliable way to obtain accurate transgene expression for in vivo studies of gene expression and function. A rate-limiting step in use of this technology to characterize large numbers of genes has been the process with which BACs can be modified by homologous recombination in Escherichia coli. We report here a highly efficient method for modifying BACs by using a novel set of shuttle vectors that contain the R6Kγ origin for DNA replication, the E. coli RecA gene for recombination, and the SacBgene for negative selection. These new vectors greatly increased the ease with which one can clone the shuttle vectors, as well as screen for co-integrated and resolved clones. Furthermore, we simplify the shuttle vector cloning to one step by incorporation of a “built-in” resolution cassette for rapid removal of the unwanted vector sequences. This new system has been used to modify a dozen BACs. It is well suited for efficient production of modified BACs for use in a variety of in vivo studies.

Mutations in PTEN-induced kinase 1 (PINK1), a mitochondrial Ser/ Thr kinase, cause an autosomal recessive form of Parkinson's disease (PD), PARK6. To investigate the mechanism of PINK1 pathogenesis, we used the Drosophila Pink1 knockout (KO) model. In mitochondria isolated from Pink1-KO flies, mitochondrial respiration driven by the electron transport chain (ETC) is significantly reduced. This reduction is the result of a decrease in ETC complex I and IV enzymatic activity. As a consequence, Pink1-KO flies also display a reduced mitochondrial ATP synthesis. Because mitochondrial dynamics is important for mitochondrial function and Pink1-KO flies have defects in mitochondrial fission, we explored whether fission machinery deficits underlie the bioenergetic defect in Pink1-KO flies. We found that the bioenergetic defects in the Pink1-KO can be ameliorated by expression of Drp1, a key molecule in mitochondrial fission. Further investigation of the ETC complex integrity in wild type, Pink1-KO, PInk1-KO/Drp1 transgenic, or Drp1 transgenic flies indicates that the reduced ETC complex activity is likely derived from a defect in the ETC complex assembly, which can be partially rescued by increasing mitochondrial fission. Taken together, these results suggest a unique pathogenic mechanism of PINK1 PD: The loss of PINK1 impairs mitochondrial fission, which causes defective assembly of the ETC complexes, leading to abnormal bioenergetics.pathology | mitochondrial movement A mple evidence indicates that mitochondrial dysfunction plays a pivotal role in the development of Parkinson's disease (PD) (1-6). A 30-40% reduction of mitochondrial electron transport chain (ETC) complex I activity was observed in the postmortem brains of idiopathic PD patients (7-11). 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and rotenone, inhibitors of ETC complex I, induce clinical and pathological manifestations that recapitulate cardinal PD symptoms in humans and in animal models (4,(12)(13)(14)(15)(16), supporting the hypothesis that mitochondrial bioenergetic defects contribute to PD pathogenesis.The significance of mitochondrial dysfunction in the development of PD was further strengthened by the discovery of PINK1 as the causal gene of PARK6. PINK1 encodes a mitochondrial kinase (17), but its physiological role remains to be elucidated. Reduction or loss of PINK1 causes bioenergetic deficits that include loss of membrane potential, calcium buffering, ATP synthesis rate, and respiration in cell culture systems (18-21). In Drosophila and mouse PINK1-KOs, decreased ETC complex I mediated respiration and ATP content have been reported (22)(23)(24).ETC complex assembly depends on inner mitochondrial membrane integrity, which is maintained by fusion and fission processes. Fusion and fission regulate the number, size, and morphology of mitochondria in a dynamic manner, and perturbing these processes could affect membrane stability (25). Several key molecules that regulate these delicate processes have been identified: Dynamin-like GTPase (D...