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.

By applying the mammalian codon usage to Cre recombinase, we improved Cre expression, as determined by immunoblot and functional analysis, in three different mammalian cell lines. The improved Cre (iCre) gene was also designed to reduce the high CpG content of the prokaryotic coding sequence, thereby reducing the chances of epigenetic silencing in mammals. Transgenic iCre expressing mice were obtained with good frequency, and in these mice loxP-mediated DNA recombination was observed in all cells expressing iCre. Moreover, iCre fused to two estrogen receptor hormone binding domains for temporal control of Cre activity could also be expressed in transgenic mice. However, Cre induction after administration of tamoxifen yielded only low Cre activity. Thus, whereas efficient activation of Cre fusion proteins in the brain needs further improvements, our studies indicate that iCre should facilitate genetic experiments in the mouse.

Local inhibitory circuits are thought to shape neuronal information processing in the central nervous system, but it remains unclear how specific properties of inhibitory neuronal interactions translate into behavioral performance. In the olfactory bulb, inhibition of mitral/tufted cells via granule cells may contribute to odor discrimination behavior by refining neuronal representations of odors. Here we show that selective deletion of the AMPA receptor subunit GluA2 in granule cells boosted synaptic Ca(2+) influx, increasing inhibition of mitral cells. On a behavioral level, discrimination of similar odor mixtures was accelerated while leaving learning and memory unaffected. In contrast, selective removal of NMDA receptors in granule cells slowed discrimination of similar odors. Our results demonstrate that inhibition of mitral cells controlled by granule cell glutamate receptors results in fast and accurate discrimination of similar odors. Thus, spatiotemporally defined molecular perturbations of olfactory bulb granule cells directly link stimulus similarity, neuronal processing time, and discrimination behavior to synaptic inhibition.

A majority of current disease-modifying therapeutic approaches for age-related neurodegenerative diseases target their characteristic proteopathic lesions (α-synuclein, Tau, Aβ). To monitor such treatments, fluid biomarkers reflecting the underlying disease process are crucial. We found robust increases of neurofilament light chain (NfL) in CSF and blood in murine models of α-synucleinopathies, tauopathy, and β-amyloidosis. Blood and CSF NfL levels were strongly correlated, and NfL increases coincided with the onset and progression of the corresponding proteopathic lesions in brain. Experimental induction of α-synuclein lesions increased CSF and blood NfL levels, while blocking Aβ lesions attenuated the NfL increase. Consistently, we also found NfL increases in CSF and blood of human α-synucleinopathies, tauopathies, and Alzheimer's disease. Our results suggest that CSF and particularly blood NfL can serve as a reliable and easily accessible biomarker to monitor disease progression and treatment response in mouse models and potentially in human proteopathic neurodegenerative diseases.

Recent studies have shown that genetic deficiency of the adipocyte fatty acid-binding protein (aP2) results in minor alterations of plasma lipids and adipocyte development but provides significant protection from dietary obesity-induced hyperinsulinemia and insulin resistance. To identify potential mechanisms responsible for this phenotype, we examined lipolysis and insulin secretion in aP2-/- mice. Beta-adrenergic stimulation resulted in a blunted rise of blood glycerol levels in aP2-/- compared with aP2+/+ mice, suggesting diminished lipolysis in aP2-/- adipocytes. Confirming this, primary adipocytes isolated from aP2-/- mice showed attenuated glycerol and free fatty acid (FFA) release in response to dibutyryl cAMP. The decreased lipolytic response seen in the aP2-/- mice was not associated with altered expression levels of hormone-sensitive lipase or perilipin. The acute insulin secretory response to beta-adrenergic stimulation was also profoundly suppressed in aP2-/- mice despite comparable total concentrations and only minor changes in the composition of systemic FFAs. To address whether levels of specific fatty acids are different in aP2-/- mice, the plasma FFA profile after beta-adrenergic stimulation was determined. Significant reduction in both stearic and cis-11-eicoseneic acids and an increase in palmitoleic acid were observed. The response of aP2-/- mice to other insulin secretagogues such as arginine and glyburide was similar to that of aP2+/+ mice, arguing against generally impaired function of pancreatic beta-cells. Finally, no aP2 expression was detected in isolated pancreatic islet cells. These results provide support for the existence of an adipo-pancreatic axis, the proper action of which relies on the presence of aP2. Consequently, aP2's role in the pathogenesis of type 2 diabetes might involve regulation of both hyperinsulinemia and insulin resistance through its impact on both lipolysis and insulin secretion.

No abstract

Neuronal network dysfunction is increasingly recognized as an early symptom in Alzheimer's disease (AD) and may provide new entry points for diagnosis and intervention. Here, we show that amyloid-beta-induced hyperexcitability of hippocampal inhibitory parvalbumin (PV) interneurons importantly contributes to neuronal network dysfunction and memory impairment in APP/PS1 mice, a mouse model of increased amyloidosis. We demonstrate that hippocampal PV interneurons become hyperexcitable at~16 weeks of age, when no changes are observed yet in the intrinsic properties of pyramidal cells. This hyperexcitable state of PV interneurons coincides with increased inhibitory transmission onto hippocampal pyramidal neurons and deficits in spatial learning and memory. We show that treatment aimed at preventing PV interneurons from becoming hyperexcitable is sufficient to restore PV interneuron properties to wild-type levels, reduce inhibitory input onto pyramidal cells, and rescue memory deficits in APP/PS1 mice. Importantly, we demonstrate that early intervention aimed at restoring PV interneuron activity has long-term beneficial effects on memory and hippocampal network activity, and reduces amyloid plaque deposition, a hallmark of AD pathology. Taken together, these findings suggest that early treatment of PV interneuron hyperactivity might be clinically relevant in preventing memory decline and delaying AD progression.

BackgroundAlzheimer’s disease (AD) is the most common form of dementia, the number of affected individuals is rising, with significant impacts for healthcare systems. Current symptomatic treatments delay, but do not halt, disease progression. Genetic evidence points to aggregation and deposition of amyloid-β (Aβ) in the brain being causal for the neurodegeneration and dementia typical of AD. Approaches to target Aβ via inhibition of γ-secretase or passive antibody therapy have not yet resulted in substantial clinical benefits. Inhibition of BACE1 (β-secretase) has proven a challenging concept, but recent BACE1inhibitors can enter the brain sufficiently well to lower Aβ. However, failures with the first clinical BACE1 inhibitors have highlighted the need to generate compounds with appropriate efficacy and safety profiles, since long treatment periods are expected to be necessary in humans.ResultsTreatment with NB-360, a potent and brain penetrable BACE-1 inhibitor can completely block the progression of Aβ deposition in the brains of APP transgenic mice, a model for amyloid pathology. We furthermore show that almost complete reduction of Aβ was achieved also in rats and in dogs, suggesting that these findings are translational across species and can be extrapolated to humans. Amyloid pathology may be an initial step in a complex pathological cascade; therefore we investigated the effect of BACE-1 inhibition on neuroinflammation, a prominent downstream feature of the disease. NB-360 stopped accumulation of activated inflammatory cells in the brains of APP transgenic mice. Upon chronic treatment of APP transgenic mice, patches of grey hairs appeared.ConclusionsIn a rapidly developing field, the data on NB-360 broaden the chemical space and expand knowledge on the properties that are needed to make a BACE-1 inhibitor potent and safe enough for long-term use in patients. Due to its excellent brain penetration, reasonable oral doses of NB-360 were sufficient to completely block amyloid-β deposition in an APP transgenic mouse model. Data across species suggest similar treatment effects can possibly be achieved in humans. The reduced neuroinflammation upon amyloid reduction by NB-360 treatment supports the notion that targeting amyloid-β pathology can have beneficial downstream effects on the progression of Alzheimer’s disease.