Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson's disease (PD), but the underlying pathophysiological mechanisms and the normal function of this large multidomain protein remain speculative. To address the role of this protein in vivo, we generated three different LRRK2 mutant mouse lines. Mice completely lacking the LRRK2 protein (knock-out, KO) showed an early-onset (age 6 weeks) marked increase in number and size of secondary lysosomes in kidney proximal tubule cells and lamellar bodies in lung type II cells. Mice expressing a LRRK2 kinase-dead (KD) mutant from the endogenous locus displayed similar early-onset pathophysiological changes in kidney but not lung. KD mutants had dramatically reduced full-length LRRK2 protein levels in the kidney and this genetic effect was mimicked pharmacologically in wild-type mice treated with a LRRK2-selective kinase inhibitor. Knock-in (KI) mice expressing the G2019S PD-associated mutation that increases LRRK2 kinase activity showed none of the LRRK2 protein level and histopathological changes observed in KD and KO mice. The autophagy marker LC3 remained unchanged but kidney mTOR and TCS2 protein levels decreased in KD and increased in KO and KI mice. Unexpectedly, KO and KI mice suffered from diastolic hypertension opposed to normal blood pressure in KD mice. Our findings demonstrate a role for LRRK2 in kidney and lung physiology and further show that LRRK2 kinase function affects LRRK2 protein steady-state levels thereby altering putative scaffold/GTPase activity. These novel aspects of peripheral LRRK2 biology critically impact ongoing attempts to develop LRRK2 selective kinase inhibitors as therapeutics for PD.

Abstract-The aim of this study was to explore the effects of the renin inhibitor aliskiren in streptozotocin-diabetic TG(mRen-2)27 rats. Furthermore, we investigated in vitro the effect of aliskiren on the interactions between renin and the (pro)renin receptor and between aliskiren and prorenin. Aliskiren distributed extensively to the kidneys of normotensive (non)diabetic rats, localizing in the glomeruli and vessel walls after 2 hours exposure. In diabetic TG(mRen-2)27 rats, aliskiren (10 or 30 mg/kg per day, 10 weeks) lowered blood pressure, prevented albuminuria, and suppressed renal transforming growth factor-␤ and collagen I expression versus vehicle. Aliskiren reduced (pro)renin receptor expression in glomeruli, tubules, and cortical vessels compared to vehicle (in situ hybridization). In human mesangial cells, aliskiren (0.1 mol/L to 10 mol/L) did not inhibit binding of 125 I-renin to the (pro)renin receptor, nor did it alter the activation of extracellular signal-regulated kinase 1/2 by renin (20 nmol/L) preincubated with aliskiren (100 nmol/L) or affect gene expression of the (pro)renin receptor. Evidence was obtained that aliskiren binds to the active site of prorenin. The above results demonstrate the antihypertensive and renoprotective effects of aliskiren in experimental diabetic nephropathy. The evidence that aliskiren can reduce in vivo gene expression for the (pro)renin receptor and that it may block prorenin-induced angiotensin generation supports the need for additional work to reveal the mechanism of the observed renoprotection by this renin inhibitor. Key Words: aliskiren Ⅲ renin inhibitor Ⅲ TG(mRen-2)rat Ⅲ diabetic nephropathy Ⅲ (pro)renin receptor A central role for the renin-angiotensin-aldosterone system (RAAS) in the pathogenesis of diabetic nephropathy (DN) is widely accepted, based largely on the attenuation of DN by angiotensin (Ang) converting enzyme inhibitors (ACEi) 1 and Ang II receptor blockers (ARB). 2 However, these agents do not halt renal decline, possibly because of insufficient suppression of the intrarenal RAAS. Theoretically, agents that more effectively suppress the RAAS should confer improved tissue protection over current treatments for DN. Renin inhibitors, by acting at the point of activation of the RAAS cascade, may represent such agents. Aliskiren is a potent inhibitor of human renin; it lowers blood pressure (BP) in patients with mild-moderate hypertension 3,4 and shows cardiorenal protection in hypertensive double transgenic rats expressing human genes for renin and angiotensinogen. 5

gamma-Aminobutyric acidA (GABAA) receptors in the mammalian central nervous system (CNS) are members of a family of ligand-gated ion channels consisting of heterooligomeric glycoprotein complexes in synaptic and extrasynaptic membranes. Although molecular cloning studies have identified 5 subunits (with approximately 40% amino acid homology) and isoforms thereof (approximately 70% homology), namely alpha 1-6, beta 1-4, gamma 1-3, delta, and rho, the subunit composition and stoichiometry of native receptors are not known. The regional distribution and cellular expression of GABAA receptor messenger RNAs (mRNAs) in the rat CNS have now been investigated by in situ hybridization histochemistry with subunit-specific 35S-labelled oligonucleotide probes on adjacent cryostat sections. Whereas alpha 1, beta 2, and gamma 2 transcripts were the most abundant and ubiquitous in the rat brain--correlating with the radioautographic distribution of GABAA receptors revealed by an ionophore ligand--others had a more restricted expression while often being abundant. For example, alpha 2 transcripts were found only in the olfactory bulb, cerebral cortex, caudate putamen, hippocampal formation, and certain lower brain stem nuclei; alpha 3 only in the olfactory bulb and cerebral cortex; alpha 5 in the hippocampal formation; and alpha 6 only in cerebellar granule cells. In addition, beta 1, beta 3, gamma 1, and delta mRNAs were also uniquely expressed in restricted brain regions. Moreover, in the spinal cord, alpha 1-3, beta 2,3, and gamma 2 mRNAs were differently expressed in Rexed layers 2-9, with alpha 2, beta 3, and gamma 2 transcripts most prominent in motoneurons of layer 9. Although differential protein trafficking could lead to the incorporation of some subunits into somatic membranes and others into dendritic membranes, some tentative conclusions as to the probable composition of native proteins in various regions of the CNS may be drawn.(ABSTRACT TRUNCATED AT 400 WORDS)

FTY720[2-amino-2-[2-(4-octylphenyl)ethyl]propane-1,3-diol hydrochloride] is an oral sphingosine-1-phosphate receptor modulator under development for the treatment of multiple sclerosis (MS). The drug is phosphorylated in vivo by sphingosine kinase 2 to its bioactive form, FTY720-P. Although treatment with FTY720 is accompanied by a reduction of the peripheral lymphocyte count, its efficacy in MS and experimental autoimmune encephalomyelitis (EAE) may be due to additional, direct effects in the central nervous system (CNS). We now show that FTY720 localizes to the CNS white matter, preferentially along myelin sheaths. Brain trough levels of FTY720 and FTY720-P in rat EAE are of the same magnitude and dose dependently increase; they are in the range of 40 to 540 ng/g in the brain tissue at efficacious doses and exceed blood concentrations severalfold. In a rat model of chronic EAE, prolonged treatment with 0.03 mg/kg was efficacious, but limiting the dosing period failed to prevent EAE despite a significant decrease in blood lymphocytes. FTY720 effectiveness is likely due to a culmination of mechanisms involving reduction of autoreactive T cells, neuroprotective influence of FTY720-P in the CNS, and inhibition of inflammatory mediators in the brain.FTY720 is an oral sphingosine-1-phosphate (S1P) receptor modulator (Baumruker et al., 2007) under development for the treatment of multiple sclerosis (MS), representing the first of a new class of immunomodulatory agents. Promising results in phase II trials with relapsing MS patients mirror the striking efficacy of FTY720 in MS models of experimental autoimmune encephalomyelitis (EAE), shown by preventive and therapeutic treatment (Brinkmann et al., 2002;Fujino et al., 2003;Webb et al., 2004;Kataoka et al., 2005;Balatoni et al., 2007). FTY720 is converted in vivo to its biologically active phosphate ester metabolite (FTY720-P), which acts as a high-affinity agonist for four of the five known G-protein-coupled S1P receptors, namely S1P 1 and S1P 3-5 (Brinkmann et al., 2002;Mandala et al., 2002). Sphingosine kinase (SPHK) 2 is the primary enzyme required for FTY720-P formation, as we and others subsequently confirmed in SPHK2 knockout mice (Kharel et al., 2005;Zemann et al., 2006). The fact that SPHK1 null mice become lymphopenic after FTY720 administration further supports the view that SPHK2 is sufficient for the functional activation of FTY720 (Allende et al., 2004).Emerging evidence suggests that the effectiveness of FTY720 in the central nervous system (CNS) extends beyond immunomodulation to encompass other aspects of MS pathophysiology, including an influence on the blood-brain barrier and glial repair mechanisms that could ultimately contribute to restoration of nerve function (Baumruker et al., 2007; This work was supported by Novartis Pharma AG. 1

Abstract.Adhesion molecule on glia (AMOG) is a novel neural cell adhesion molecule that mediates neuron-astrocyte interaction in vitro. In situ AMOG is expressed in the cerebellum by glial cells at the critical developmental stages of granule neuron migration. Granule neuron migration that is guided by surface contacts between migrating neurons and astroglial processes is inhibited by monoclonal AMOG antibody, probably by disturbing neuron-glia adhesion. AMOG is an integral cell surface glycoprotein of 45-50-kD molecular weight with a carbohydrate content of at least 30%. It does not belong to the L2/HNK-1 family of neural cell adhesion molecules but expresses another carbohydrate epitope that is shared with the adhesion molecules L1 and myelin-associated glycoprotein, but is not present on N-CAM or J1.

In order to investigate the expression of cell adhesion molecules in synapses, we have studied the localization of the neural cell adhesion molecule N-CAM in the cerebellum and hippocampus of adult mice by immunocytological and immunochemical methods. Of the three molecular components of N-CAM with relative molecular masses (Mr) of 120, 140, and 180 kD, N-CAM 120 is not detectable in synaptosomal membranes, whereas N-CAM 140 is expressed on both pre- and postsynaptic membranes and N-CAM 180 is restricted to postsynaptic sites, with localization of the N-CAM 180-specific epitope in postsynaptic densities. Specificity of immunoreactivity is indicated by the observation that antibodies to the neural cell adhesion molecule L1 do not label synaptic membranes, whereas antibodies to two major components of postsynaptic densities, actin and erythrocyte spectrin, react with synaptic structures. Interestingly, N-CAM 180 is only detectable in subpopulations of synapses in the intact tissue. Isolated synaptosomes, opened for unimpeded accessibility of antibody by hypoosmotic treatment, also reveal a partial expression of N-CAM 180 in that 67% are labeled by antibodies to N-CAM 180, while antibodies to actin and erythrocyte spectrin react with 95% and 88% of all synaptosomes, respectively. N-CAM 180 does not appear to be differentially expressed in synapses of a particular morphological type, but is detectable in all types of synapses in the cerebellum and hippocampus, except for mossy fiber synapses and synapses between basket and Purkinje cells, which are generally N-CAM 180-negative. Since N-CAM 180 has been shown to be characteristic of stabilized or stabilizing cell contacts, possibly by its association with the cytoskeleton-membrane linker protein spectrin (Pollerberg et al.: J. Cell Biol. 101:1921-1929, '85; Nature 324:462-465, '86; Cell Tissue Res. 250:227-236, '87), we would like to suggest N-CAM 180 plays an important role in determining the stability of contacts between pre- and postsynaptic membranes and state of synaptic activity.

Abstract. The cellular and subcellular localization of the neural cell adhesion molecules L1 and N-CAM was studied by pre-and postembedding immunoelectron microscopic labeling procedures in the developing mouse cerebellar cortex. The salient features of the study are: (a) L1 displays a previously unrecognized restricted expression by particular neuronal cell types (i.e., it is expressed by granule cells but not by stellate and basket cells) and by particular subcellular compartments (i.e., it is expressed on axons but not on dendrites or cell bodies of Purkinje cells). (b) L1 is always expressed on fasciculating axons and on postmitotic, premigratory, and migrating granule cells at sites of neuron-neuron contact, but never at contact sites between neuron and glia, thus strengthening the view that L1 is not involved in granule cell migration as a neuron-glia adhesion molecule. (c) While N-CAM antibodies reacting with the three major components of N-CAM (180, 140, and 120 kD) show a rather uniform labeling of all cell types, antibodies to the 180-kD component (N-CAM1s0) stain only the postmigratory granule cell bodies supporting the notion that N-CAMps0, the N-CAM component with the longest cytoplasmic domain, is not expressed before stable cell contacts are formed. Furthermore, N-CAMls0 is only transiently expressed on Purkinje cell dendrites. (d) N-CAM is present in synapses on both pre-and postsynaptic membranes. L1 is expressed only preterminally and not in the subsynaptic membranes.These observations indicate an exquisite degree of fine tuning in adhesion molecule expression during neural development and suggest a rich combinatorial repertoire in the specification of cell surface contacts.

e Brown adipose tissue (BAT) is a key tissue for energy expenditure via fat and glucose oxidation for thermogenesis. In this study, we demonstrate that the myostatin/activin receptor IIB (ActRIIB) pathway, which serves as an important negative regulator of muscle growth, is also a negative regulator of brown adipocyte differentiation. In parallel to the anticipated hypertrophy of skeletal muscle, the pharmacological inhibition of ActRIIB in mice, using a neutralizing antibody, increases the amount of BAT without directly affecting white adipose tissue. Mechanistically, inhibition of ActRIIB inhibits Smad3 signaling and activates the expression of myoglobin and PGC-1 coregulators in brown adipocytes. Consequently, ActRIIB blockade in brown adipose tissue enhances mitochondrial function and uncoupled respiration, translating into beneficial functional consequences, including enhanced cold tolerance and increased energy expenditure. Importantly, ActRIIB inhibition enhanced energy expenditure only at ambient temperature or in the cold and not at thermoneutrality, where nonshivering thermogenesis is minimal, strongly suggesting that brown fat activation plays a prominent role in the metabolic actions of ActRIIB inhibition.