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Parkinson's disease (PD) is a progressive neurodegenerative disease caused by the interaction of genetic and environmental factors. However, the etiology of PD remains largely unknown. Macroautophagy is known to play an essential role in the degradation of abnormal proteins and organelles. Furthermore, the loss of autophagy-related (Atg) genes results in neurodegeneration and abnormal protein accumulation. Since these are also pathologic features of Parkinson's disease, the conditional impairment of autophagy may lead to improved animal models for the study of PD. Using transgenic mice expressing Cre recombinase under the control of either the dopamine transporter or the engrailed-1 promoters, we generated mice with the conditional deletion of Atg7 in the dopamine neurons of the substantia nigra pars compacta, other regions of the midbrain, and also the hindbrain. This conditional impairment of autophagy results in the age-related loss of dopaminergic neurons and corresponding loss of striatal dopamine, the accumulation of low-molecular-weight α-synuclein, and the presence of ubiquitinated protein aggregates, recapitulating many of the pathologic features of PD. These conditional knock-out animals provide insight into the process of autophagy in Parkinson's disease pathology.

Maintaining adequate numbers of spermatogonial stem cells is required for the production of the millions of sperm required for male fertility. To date, however, the mechanisms that regulate the size of this pool in the adult are poorly defined. Glial cell line-derived neurotrophic factor (GDNF) is required for establishing this pool in the prepubertal animal, but its in vivo function in the normal adult testis has never been examined directly. We used a chemical-genetic approach to address this issue. We generated mice carrying a single amino acid mutation (V805A) in Ret, the kinase subunit of the GDNF receptor. This mutation does not affect normal GDNF signaling but renders it susceptible to inhibition by the ATP competitive inhibitor, NA-PP1. When GDNF signaling was blocked in adults for 11 days, only a few cells remained that expressed the stem spermatogonial markers, Gfrα1 and Zbtb16, and testicular Ret mRNA content was reduced markedly. These decreases were associated with depletion of functional stem spermatogonia; some were lost when GDNF signaling was inhibited for only 2 days while others survived for up to 11 days. However, when signaling was restored, the remaining stem cells proliferated, initiating tissue restoration. In conclusion, these results provide the first direct proof that GDNF acutely regulates the number of spermatogonial stem cells in the normal adult testis. Additionally, these results demonstrate different sensitivities among subpopulation of these stem cells to inhibition of GDNF signaling. Disclosure of potential conflicts of interest is found at the end of this article.

Inhibitors of apoptosis (IAPs) are a family of highly-conserved proteins that regulate cell survival through binding to caspases, the final executioners of apoptosis. X-linked IAP (XIAP) is the most widely expressed IAP and plays an important function in regulating cell survival. XIAP contains 3 baculoviral IAP repeats (BIRs) followed by a RING finger domain at the C terminal. The BIR domains of XIAP possess anticaspase activities, whereas the RING finger domain enables XIAP to function as an E3 ubiquitin ligase in the ubiquitin and proteasomal system. Our previous study showed that parkin, a protein that is important for the survival of dopaminergic neurons in Parkinson's disease (PD), is S-nitrosylated both in vitro and in vivo in PD patients. S-nitrosylation of parkin compromises its ubiquitin E3 ligase activity and its protective function, which suggests that nitrosative stress is an important factor in regulating neuronal survival during the pathogenesis of PD. In this study we show that XIAP is S-nitrosylated in vitro and in vivo in an animal model of PD and in PD patients. Nitric oxide modifies mainly cysteine residues within the BIR domains. In contrast to parkin, S-nitrosylation of XIAP does not affect its E3 ligase activity, but instead directly compromises its anticaspase-3 and antiapoptotic function. Our results confirm that nitrosative stress contributes to PD pathogenesis through the impairment of prosurvival proteins such as parkin and XIAP through different mechanisms, indicating that abnormal S-nitrosylation plays an important role in the process of neurodegeneration.

NGF controls survival, differentiation, and target innervation of both peptidergic and nonpeptidergic DRG sensory neurons. The common receptor for GDNF family ligands, Ret, is highly expressed in nonpeptidergic neurons, but its function during development of these neurons is unclear. Here, we show that expression of Ret and its coreceptors GFRalpha1 and GFRalpha2 is dependent on NGF. GFR/Ret signaling, in turn, autoregulates expression of both GFRalpha1 and GFRalpha2 and promotes expression of TrpA1, MrgA1, MrgA3, and MrgB4, acquisition of normal neuronal size, axonal innervation of the epidermis, and postnatal extinction of the NGF receptor TrkA. Moreover, NGF controls expression of several other genes characteristic of nonpeptidergic neurons, such as TrpC3, TrpM8, MrgD, and the transcription factor Runx1, via a Ret-independent signaling pathway. These findings support a model in which NGF controls maturation of nonpeptidergic DRG neurons through a combination of GFR/Ret-dependent and -independent signaling pathways.

RET can be activated in cis or trans by its co-receptors and ligands in vitro, but the physiological roles of trans signaling are unclear. Rapidly adapting (RA) mechanoreceptors in dorsal root ganglia (DRGs) express Ret and the co-receptor Gfrα2 and depend on Ret for survival and central projection growth. Here, we show that Ret and Gfrα2 null mice display comparable early central projection deficits, but Gfrα2 null RA mechanoreceptors recover later. Loss of Gfrα1, the co-receptor implicated in activating RET in trans, causes no significant central projection or cell survival deficit, but Gfrα1;Gfrα2 double nulls phenocopy Ret nulls. Finally, we demonstrate that GFRα1 produced by neighboring DRG neurons activates RET in RA mechanoreceptors. Taken together, our results suggest that trans and cis RET signaling could function in the same developmental process and that the availability of both forms of activation likely enhances but not diversifies outcomes of RET signaling. DOI: http://dx.doi.org/10.7554/eLife.06828.001, eLife digest During development, cells send and receive numerous signaling molecules. In order to trigger a biological response, such signaling molecules must first bind to a specific receptor protein, often located on the cell surface. These receptor proteins can either work alone or with partner proteins called co-receptors. When the co-receptor is produced by the same cell as the receptor, it is called cis signaling. When the co-receptor is produced by other cells, it is called trans signaling. RET is one such receptor that is important for the development of the nervous system and many other biological processes. It interacts with a particular family of signaling molecules, the glial cell line-derived neurotrophic factor (GDNF) family ligands, which first bind to a co-receptor, GFRα, before binding to RET. These co-receptors can come from the same cell as RET, or from a different cell. Previous studies have indicated that RET can receive both cis and trans signals using cultured cells, but it was not clear whether both types of signal occur during normal development and contribute to the same biological processes. Fleming, Vysochan et al. investigated this question by analyzing the roles of RET signaling in a type of mouse neuron that is involved in sensing touch. RET is important for the survival and development of these neurons, which express both RET and its co-receptor GFRa2. Another RET co-receptor, GFRa1, is produced by other cells that are next to the cell bodies and projections of these touch-sensing neurons. To investigate the roles of different GFRa co-receptors further, Fleming, Vysochan et al. generated a variety of mouse mutants, including mice with mutations in one or both types of co-receptor. The neurons in mice lacking both co-receptors shared the same defects as the neurons in the mice lacking RET. Loss of either co-receptor alone did not produce these abnormalities. This indicates that both co-receptors can mediate the normal development of these neurons, with GFRa2 signaling in cis and GFRa1 signaling in trans. Fleming, Vysochan et al. propose that cis and trans RET signaling can lead to the same biological outcomes in these neurons. Future experiments should reveal if cis and trans RET signaling contribute towards common biological processes in other cell types inside the body as well. Such findings might also be important for understanding the role of RET signaling in cancer and other human diseases. DOI: http://dx.doi.org/10.7554/eLife.06828.002

Mutations in the genes coding for alpha-synuclein and parkin cause autosomal-dominant and autosomal-recessive forms of Parkinson's disease (PD), respectively. Alpha-synuclein is a major component of Lewy bodies, the proteinaceous cytoplasmic inclusions that are the pathological hallmark of idiopathic PD. Lewy bodies appear to be absent in cases of familial PD associated with mutated forms of parkin. Parkin is an ubiquitin E3 ligase, and it may be involved in the processing and/or degradation of alpha-synuclein, as well as in the formation of Lewy bodies. Here we report the behavioral, biochemical, and histochemical characterization of double-mutant mice overexpressing mutant human A53T alpha-synuclein on a parkin null background. We find that the absence of parkin does not have an impact on the onset and progression of the lethal phenotype induced by overexpression of human A53T alpha-synuclein. Furthermore, all major behavioral, biochemical, and morphological characteristics of A53T alpha-synuclein-overexpressing mice are not altered in parkin null alpha-synuclein-overexpressing double-mutant mice. Our results demonstrate that mutant alpha-synuclein induces neurodegeneration independent of parkin-mediated ubiquitin E3 ligase activity in nondopaminergic systems and suggest that PD caused by alpha-synuclein and parkin mutations may occur via independent mechanisms.

The nontoxic C fragment of tetanus toxin (TC) can transport other proteins from the circulation to central nervous system (CNS) motor neurons. Increased levels of CuZn superoxide dismutase (SOD) are protective in experimental models of stroke and Parkinson's disease, whereas mutations in SOD can cause motor neuron disease. We have linked TC to SOD and purified the active recombinant proteins in both the TC-SOD and SOD-TC orientations. Light microscopic immunohistochemistry and quantitative enzyme-linked immunosorbant assays (ELISA) of mouse brainstem, after intramuscular injection, demonstrate that the fusion proteins undergo retrograde axonal transport and transsynaptic transfer as efficiently as TC alone.

Ubiquitous classical (typical) calpains, calpain-1 and calpain-2, are Ca+2-dependent cysteine proteases, which have been associated with numerous physiological and pathological cellular functions. However, a clear understanding of the role of calpains in the CNS has been hampered by the lack of appropriate deletion paradigms in the brain. In this study, we describe a unique model of conditional deletion of both calpain-1 and calpain-2 activities in mouse brain, which more definitively assesses the role of these ubiquitous proteases in brain development/function and pathology. Surprisingly, we show that these calpains are not critical for gross CNS development. However, calpain-1/calpain-2 loss leads to reduced dendritic branching complexity and spine density deficits associated with major deterioration in hippocampal long-term potentiation and spatial memory. Moreover, calpain-1/calpain-2-deficient neurons were significantly resistant to injury induced by excitotoxic stress or mitochondrial toxicity. Examination of downstream target showed that the conversion of the Cdk5 activator, p35, to pathogenic p25 form, occurred only in the presence of calpain and that it played a major role in calpain-mediated neuronal death. These findings unequivocally establish two central roles of calpain-1/calpain-2 in CNS function in plasticity and neuronal death.

Ghrelin targets the hypothalamus to regulate food intake and adiposity. Endogenous ghrelin receptors [growth hormone secretagogue receptor (GHSR)] are also present in extrahypothalamic sites where they promote circuit activity associated with learning and memory, and reward seeking behavior. Here, we show that the substantia nigra pars compacta (SNpc), a brain region where dopamine (DA) cell degeneration leads to Parkinson's disease (PD), expresses GHSR. Ghrelin binds to SNpc cells, electrically activates SNpc DA neurons, increases tyrosine hydroxylase mRNA and increases DA concentration in the dorsal striatum. Exogenous ghrelin administration decreased SNpc DA cell loss and restricted striatal dopamine loss after 1-methyl-4-phenyl-1,2,5,6 tetrahydropyridine (MPTP) treatment. Genetic ablation of ghrelin or the ghrelin receptor (GHSR) increased SNpc DA cell loss and lowered striatal dopamine levels after MPTP treatment, an effect that was reversed by selective reactivation of GHSR in catecholaminergic neurons. Ghrelin-induced neuroprotection was dependent on the mitochondrial redox state via uncoupling protein 2 (UCP2)-dependent alterations in mitochondrial respiration, reactive oxygen species production, and biogenesis. Together, our data reveal that peripheral ghrelin plays an important role in the maintenance and protection of normal nigrostriatal dopamine function by activating UCP2-dependent mitochondrial mechanisms. These studies support ghrelin as a novel therapeutic strategy to combat neurodegeneration, loss of appetite and body weight associated with PD. Finally, we discuss the potential implications of these studies on the link between obesity and neurodegeneration.

Objective The PARK8 gene responsible for late-onset autosomal dominant Parkinson's disease encodes a large novel protein of unknown biological function termed leucine-rich repeat kinase 2 (LRRK2). The studies herein explore the localization of LRRK2 in the mammalian brain. Methods Polyclonal antibodies generated against the amino or carboxy termini of LRRK2 were used to examine the biochemical, subcellular, and immunohistochemical distribution of LRRK2. Results LRRK2 is detected in rat brain as an approximate 280kDa protein by Western blot analysis. Subcellular fractionation demonstrates the presence of LRRK2 in microsomal, synaptic vesicle–enriched and synaptosomal cytosolic fractions from rat brain, as well as the mitochondrial outer membrane. Immunohistochemical analysis of rat and human brain tissue and primary rat cortical neurons, with LRRK2-specific antibodies, shows widespread neuronal-specific labeling localized exclusively to punctate structures within perikarya, dendrites, and axons. Confocal colocalization analysis of primary cortical neurons shows partial yet significant overlap of LRRK2 immunoreactivity with markers specific for mitochondria and lysosomes. Furthermore, ultrastructural analysis in rodent basal ganglia detects LRRK2 immunoreactivity associated with membranous and vesicular intracellular structures, including lysosomes, endosomes, transport vesicles, and mitochondria. Interpretation The association of LRRK2 with a variety of membrane and vesicular structures, membrane-bound organelles, and microtubules suggests an affinity of LRRK2 for lipids or lipid-associated proteins and may suggest a potential role in the biogenesis and/or regulation of vesicular and membranous intracellular structures within the mammalian brain. Ann Neurol 2006;60:557–569

Parkinson disease (PD) is a relatively common disorder of the nervous system that afflicts patients later in life with tremor, slowness of movement, gait instability, and rigidity. Treatment of these cardinal features of the disease is a success story of modern science and medicine, as a great deal of disability can be alleviated through the pharmacological correction of brain dopamine deficiency. Unfortunately these therapies only provide temporary, though significant, relief from early symptoms and do not halt disease progression. In addition, pathological changes outside of the motor system leading to cognitive, autonomic, and psychiatric symptoms are not sufficiently treated by current therapies. Much as the discovery of dopamine deficiency led to powerful treatments for motor symptoms, recent discoveries concerning the role of specific genes in PD pathology will lead to the next revolution in disease therapy. Understanding why and how susceptible cells in motor and nonmotor regions of the brain die in PD is the first step toward preventing this cell death and curing or slowing the disease. In this review we discuss recent discoveries in the fields of diagnosis and treatment of PD and focus on how a better understanding of disease mechanisms gained through the study of monogenetic forms of PD has provided novel therapeutic targets.

Recent findings have uncovered a role for theBcl-xgene in the survival of dopaminergic neurons. The exact nature of this role has been difficult to examine because of the embryonic lethality ofBcl-xgene disruption in mouse models. Here we report the generation catecholaminergic cell-specific conditionalBcl-xgene knock-out mice using Cre-lox recombination technology. First we produced transgenic mice that express Cre recombinase from an exogenous rat tyrosine hydroxylase promoter (TH-Cremice). These mice were crossed toZ/APandZ/EGreporter mouse strains to verify catecholaminergic (TH-positive) cell-specific Cre expression. TheTH-Cremice then were mated to mice possessing theBcl-xgene flanked by loxP sites, thereby producing offspring withBcl-xdeletion limited to catecholaminergic cells. The resulting mice are viable but have one-third fewer catecholaminergic neurons than do control animals. They demonstrate a deficiency in striatal dopamine and also tend to be smaller and have decreased brain mass when compared with controls. Surprisingly, surviving neurons were found that lacked Bcl-x immunoreactivity, thereby demonstrating that this gene is dispensable for the ongoing survival of a subpopulation of catecholaminergic cells.

Parkinson's disease (PD) is the most common neurodegenerative movement disorder and is characterized pathologically by degeneration of catecholaminergic neurons of the substantia nigra pars compacta and locus coeruleus, among other regions. Autosomal-recessive juvenile Parkinsonism (ARJP) is caused by mutations in the PARK2 gene coding for parkin and constitutes the most common familial form of PD. The majority of ARJP-associated parkin mutations are thought to be loss of function-mutations; however, the pathogenesis of ARJP remains poorly understood. Here, we report the generation of parkin null mice by targeted deletion of parkin exon 7. These mice show a loss of catecholaminergic neurons in the locus coeruleus and an accompanying loss of norepinephrine in discrete regions of the central nervous system. Moreover, there is a dramatic reduction of the norepinephrine-dependent startle response. The nigrostriatal dopaminergic system does not show any impairment. This mouse model will help gain a better understanding of parkin function and the mechanisms underlying parkin-associated PD.

Tetanus toxin and its atoxic binding fragment, C-fragment (CF), are the only known proteins which undergo extensive transfer from motoneurons to presynaptic terminals in the spinal cord. Intramuscular injection of CF conjugated to horseradish peroxidase (HRP) was used to elucidate the ultrastructural basis for this unique property. Motoneurons labeled by retrograde axonal transport contained enzymatic reaction product within cytoplasmic vesicles and were surrounded by presynaptic terminals with label in the synaptic cleft and synaptic vesicles. Membranous structures on both sides of the synapse remained labeled for several days after the injection. Nonsynaptic regions were virtually free of CF-HRP. Transport of CF may reflect the movement of the tetanus toxin receptor in a unique synaptic compartment.

A close spatial relationship between serotonin-containing mast cells and substance P-containing nerves was shown by immunohistochemistry using a combination of antisera specific for serotonin and substance P. This supports earlier morphological results suggesting an innervation of mast cells and pharmacological studies which postulate an influence of substance P on the release of histamine from mast cells.

Topographically graded molecules representing position-specific differences among otherwise similar cells are thought to play a role in the patterning of the developing nervous system. In the embryonic chick visual system, a 40 kDa protein, TOPAP, is expressed in a posterior > anterior gradient in the retina and in an inverted anterior > posterior gradient in the optic tectum, the major retinal projection area. Here we report the isolation and nucleotide sequencing of a complementary DNA clone encoding the chick TOPAP protein and demonstrate that the mRNA encoding this coiled-coil integral membrane protein is topographically graded within the retina and is present in a variety of chick tissues.

Plasma proteins including immunoglobulins have been previously localized in neurons with processes extending outside the blood-brain barrier, but not within glia under normal conditions. Immune modulating functions have been proposed for both microglia and astrocytes in several pathological states. Using immunocytochemistry, we have found that large numbers of neuroglial cells contain immunoglobulin G (IgG) in normal mice of the C57 BL/6 strain. Most IgG-positive cells had both the morphology and distribution of microglia, including a higher density in grey matter, and were frequently found in perivascular or perineuronal locations. The accumulation of IgG does not appear to be by nonselective phagocytosis of extracellular fluid, since serum albumin could not be detected within microglia. There was little overlap in the distribution of cellular processes positive for IgG and those which showed astrocytic markers. Neuronal accumulation of plasma proteins was also seen in a distribution described by previous investigators. The function of this selective accumulation of IgG by normal microglia is unknown, but may reflect a role in the immune response within the central nervous system.