Caroline Kent

Impaired dopaminergic neurotransmission and microtubule-associated protein tau alterations in human LRRK2 transgenic mice.

Mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) gene, first described in 2004 have now emerged as the most important genetic finding in both autosomal dominant and sporadic Parkinsons disease (PD). While a formidable research effort has ensued since the initial gene discovery, little is known of either the normal or the pathological role of LRRK2. We have created lines of mice that express human wild-type (hWT) or G2019S Lrrk2 via bacterial artificial chromosome (BAC) transgenesis. In vivo analysis of the dopaminergic system revealed abnormal dopamine neurotransmission in both hWT and G2019S transgenic mice evidenced by a decrease in extra-cellular dopamine levels, which was detected without pharmacological manipulation. Immunopathological analysis revealed changes in localization and increased phosphorylation of microtubule binding protein tau in G2019S mice. Quantitative biochemical analysis confirmed the presence of differential phospho-tau species in G2019S mice but surprisingly, upon dephosphorylation the tau isoform banding pattern in G2019S mice remained altered. This suggests that other post-translational modifications of tau occur in G2019S mice. We hypothesize that Lrrk2 may impact on tau processing which subsequently leads to increased phosphorylation. Our models will be useful for further understanding of the mechanistic actions of LRRK2 and future therapeutic screening.

Adult neurogenesis and neurite outgrowth are impaired in LRRK2 G2019S mice

The generation and maturation of adult neural stem/progenitor cells are impaired in many neurodegenerative diseases, among them is Parkinsons disease (PD). In mammals, including humans, adult neurogenesis is a lifelong feature of cellular brain plasticity in the hippocampal dentate gyrus (DG) and in the subventricular zone (SVZ)/olfactory bulb system. Hyposmia, depression, and anxiety are early non-motor symptoms in PD. There are parallels between brain regions associated with non-motor symptoms in PD and neurogenic regions. In autosomal dominant PD, mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are frequent. LRRK2 homologs in non-vertebrate systems play an important role in chemotaxis, cell polarity, and neurite arborization. We investigated adult neurogenesis and the neurite development of new neurons in the DG and SVZ/olfactory bulb system in bacterial artificial chromosome (BAC) human Lrrk2 G2019S transgenic mice. We report that mutant human Lrrk2 is highly expressed in the hippocampus in the DG and the SVZ of adult Lrrk2 G2019S mice. Proliferation of newly generated cells is significantly decreased and survival of newly generated neurons in the DG and olfactory bulb is also severely impaired. In addition, after stereotactic injection of a GFP retrovirus, newly generated neurons in the DG of Lrrk2 G2019S mice exhibited reduced dendritic arborization and fewer spines. This loss in mature, developed spines might point towards a decrease in synaptic connectivity. Interestingly, physical activity partially reverses the decrease in neuroblasts observed in Lrrk2 G2010S mice. These data further support a role for Lrrk2 in neuronal morphogenesis and provide new insights into the role of Lrrk2 in adult neurogenesis.

LRRK2 knockout mice have an intact dopaminergic system but display alterations in exploratory and motor co-ordination behaviors

Mutations in the LRRK2 gene are the most common cause of genetic Parkinson’s disease. Although the mechanisms behind the pathogenic effects of LRRK2 mutations are still not clear, data emerging from in vitro and in vivo models suggests roles in regulating neuronal polarity, neurotransmission, membrane and cytoskeletal dynamics and protein degradation.We created mice lacking exon 41 that encodes the activation hinge of the kinase domain of LRRK2. We have performed a comprehensive analysis of these mice up to 20 months of age, including evaluation of dopamine storage, release, uptake and synthesis, behavioral testing, dendritic spine and proliferation/neurogenesis analysis.Our results show that the dopaminergic system was not functionally comprised in LRRK2 knockout mice. However, LRRK2 knockout mice displayed abnormal exploratory activity in the open-field test. Moreover, LRRK2 knockout mice stayed longer than their wild type littermates on the accelerated rod during rotarod testing. Finally, we confirm that loss of LRRK2 caused degeneration in the kidney, accompanied by a progressive enhancement of autophagic activity and accumulation of autofluorescent material, but without evidence of biphasic changes.

Septo-hippocampal cholinergic and neurotrophin markers in age-induced cognitive decline

Messenger RNA (mRNA) molecules encoding proteins related to the presynaptic cholinergic and neurotrophin systems were quantitated in the hippocampus and basal forebrain of Long-Evans rats with spatial learning ability assessed in the Morris water maze. The reverse transcriptase-polymerase chain reaction showed that the mRNAs for the low-affinity neurotrophin receptor (p75-NTR) and the growth-associated protein GAP-43 were decreased in level in the basal forebrain of aged-impaired rats. In the hippocampus of these aged-impaired rats, the mRNA for VGF, another neurotrophin-inducible gene, also was decreased. In situ hybridization histochemistry revealed that mRNAs for nerve growth factor (NGF) and brain-derived neurotrophic factor increased in level in the aged rat hippocampus; when age effects were removed, NGF mRNA level remained significantly correlated with maze performance. Enzyme-linked immunosorbent assay indicated that NGF protein was expressed at normal levels in the aged rat hippocampus. These mRNA and protein alterations may signify that a defect in neurotrophin signaling exists in the brains of aged Long-Evans rats, underlying reduced plasticity responses in the basal forebrain cholinergic system.

In vivo silencing of alpha-synuclein using naked siRNA

BackgroundOverexpression of α-synuclein (SNCA) in families with multiplication mutations causes parkinsonism and subsequent dementia, characterized by diffuse Lewy Body disease post-mortem. Genetic variability in SNCA contributes to risk of idiopathic Parkinsons disease (PD), possibly as a result of overexpression. SNCA downregulation is therefore a valid therapeutic target for PD.ResultsWe have identified human and murine-specific siRNA molecules which reduce SNCA in vitro. As a proof of concept, we demonstrate that direct infusion of chemically modified (naked), murine-specific siRNA into the hippocampus significantly reduces SNCA levels. Reduction of SNCA in the hippocampus and cortex persists for a minimum of 1 week post-infusion with recovery nearing control levels by 3 weeks post-infusion.ConclusionWe have developed naked gene-specific siRNAs that silence expression of SNCA in vivo. This approach may prove beneficial toward our understanding of the endogenous functional equilibrium of SNCA, its role in disease, and eventually as a therapeutic strategy for α-synucleinopathies resulting from SNCA overexpression.

Aging elevates metabolic gene expression in brain cholinergic neurons

The basal forebrain (BF) cholinergic system is selectively vulnerable in human brain diseases, while the cholinergic groups in the upper pons of the brainstem (BS) resist neurodegeneration. Cholinergic neurons (200 per region per animal) were laser-microdissected from five young (8 months) and five aged (24 months) F344 rats from the BF and the BS pontine lateral dorsal tegmental/pedunculopontine nuclei (LDTN/PPN) and their expression profiles were obtained. The bioinformatics program SigPathway was used to identify gene groups and pathways that were selectively affected by aging. In the BF cholinergic system, aging most significantly altered genes involved with a variety of metabolic functions. In contrast, BS cholinergic neuronal age effects included gene groupings related to neuronal plasticity and a broad range of normal cellular functions. Transcription factor GA-binding protein alpha (GABPalpha), which controls expression of nuclear genes encoding mitochondrial proteins, was more strongly upregulated in the BF cholinergic neurons (+107%) than in the BS cholinergic population (+40%). The results suggest that aging elicits elevates metabolic activity in cholinergic populations and that this occurs to a much greater degree in the BF group than in the BS group.

Expression of superoxide dismutase messenger RNA in adult rat brain cholinergic neurons.

Superoxide dismutase (SOD) protects cells exposed to an excess of the free radical nitric oxide, by preventing the formation of peroxynitrite. Certain central cholinergic neurons express constitutive nitric oxide synthase (nNOS), and presumably they are at risk from peroxynitrite intoxication. Immunocytochemistry for choline acetyltransferase (ChAT) was combined with in situ hybridization histochemistry (ISHH) to examine whether brain cholinergic populations differ with respect to their expression of the messenger RNA molecules (mRNAs) for the manganese-dependent (Mn-SOD) and copper/zinc-dependent superoxide dismutases (Cu/Zn-SOD).The cholinergic neurons located in the reticular formation of the upper brainstem (the laterodorsal tegmental nucleus [LDTN] and the pedunculopontine nucleus [PPN]) were found to express relatively high levels of Mn-SOD mRNA, whereas cholinergic neurons located in the basal forebrain (substantia innominata [SI], diagonal band [DB], medial septum [MS], and the nucleus basalis magnocellularis [nBM]), and the striatal cholinergic interneurons expressed low to intermediate levels of Mn-SOD mRNA. The rank order of median Mn-SOD mRNA density per cholinergic cell was LDTN > PPN > SI > striatum = nBM = DB > MS. This is similar to the rank order of nNOS mRNA densities in the cholinergic cells in these regions (R=0.9, p<0.02). The rank order of Cu/Zn-SOD mRNA levels in cholinergic populations (DB > LDTN = PPN = MS > SI = nBM = striatum) was not correlated with nNOS mRNA (R = 0.29, P>0.05). Thus, for cholinergic neurons, Mn-SOD may be important for protection from NO-related oxidative stress.

Pontine cholinergic neurons depend on three neuroprotection systems to resist nitrosative stress

Brainstem cholinergic populations survive in neurodegenerative disease, while basal forebrain cholinergic neurons degenerate. We have postulated that variable resistance to oxidative stress may in part explain this. Rat primary cultures were used to study the effects of several nitrosative/oxidative stressors on brainstem (upper pons, containing pedunculopontine and lateraldorsal tegmental nuclei; BS) cholinergic neurons, comparing them with medial septal (MS), and striatal cholinergic neurons. BS cholinergic neurons were significantly more resistant to S-nitro-N-acetyl-d,l-penicillamine (SNAP), sodium nitroprusside (SNP), and hydrogen peroxide than were MS cholinergic neurons, which in turn were more resistant than striatal cholinergic neurons. Pharmacological analyses using specific inhibitors of neuroprotective systems also revealed differences between these three cholinergic populations with respect to their vulnerability to SNAP. Toxicity of SNAP to BS neurons was exacerbated by blocking NF-kappaB activation with SN50 or ERK1/2 activation by PD98059, or by inhibition of phosphoinositide-3 kinase (PI3K) activity by LY294002. In contrast, SNAP toxicity to MS neurons was augmented only by SN50, and SNAP toxicity to striatal cholinergic neurons was not increased by any of these three pharmacological agents. In neuron-enriched primary cultures, BS cholinergic neurons remained resistant to SNAP while MS cholinergic neurons remained vulnerable to this agent. Immunohistochemical experiments demonstrated nitric oxide (NO)-induced increases in nuclear levels of phospho-epitopes for ERK1/2 and Akt, and of the p65 subunit of NF-kappaB, within BS cholinergic neurons. These data indicate that the relative resistance of BS cholinergic neurons to toxic levels of nitric oxide involves three intrinsic neuroprotective pathways that control transcriptional and anti-apoptotic cellular functions.

P1-108: Gene expression changes after MAPT expression inhibition

Ts65Dn mice, but not Ts1Cje, exhibited significantly reduced T2 relaxation times selectively in the medial septal area, from where BFCN originate, and in brain regions normally innervated by BFCN, including hippocampus and cingulate cortex. BFCN projections to these areas, identified by choline acetyltransferase (CHAT) immunocytochemistry or acetylcholinesterase histochemistry, were selectively and markedly reduced in Ts65Dn mice and numbers of detectable BFCN in the medial septal area, identified by CHAT or p75NGFR immunolabeling, were significantly below normal. In BFCN projection areas of the cortex, dendrites in Ts65Dn brain displayed morphological changes and markedly increased levels of MAP-2, a cytoskeletal protein regulated by cholinergic innervation. Using electron microscopy and antibody markers for apoptosis pathway activation, we detected a higher than normal frequency of neurons undergoing apoptosis in affected brain regions. Conclusions: MRI revealed more widespread neuropathology in Ts65Dn mice than previously appreciated, involving postsynaptic as well as presynaptic elements of the BFCN circuit. The cholinergic deficits, MRI T2 changes, and AD-related endosomal phenotype in Ts65Dn mice, but not Ts1Cje, support growing evidence linking APP triplication to endosome dysfunction (A.Boyer-Boiteau; et al., this meeting), retrograde signaling deficits, and increased vulnerability of neurons to degeneration.