Cefepime induced neurotoxicity: A case series and review of the literature.

Cefepime is a fourth generation cephalosporin which is bactericidal for broad spectrum of organisms. This is a case-series of three patients who presented to our hospital with confusion secondary to cefepime use to treat urinary tract infection (UTI) and health care associated pneumonia (HCAP), after excluding other common etiologies of altered mental status (AMS). Of these three patients, one had progressive expressive aphasia and the other two demonstrated asynchronous myoclonic activity of the limbs. The symptoms were seen within four to five days of initiating the treatment and resolved within three days of discontinuation of cefepime. Acute structural abnormalities were excluded by computed tomography (CT) and magnetic resonance imaging (MRI) of the brain. Electroencephalogram (EEG) showed diffuse slowing activity with triphasic waves consistent with encephalopathy. In one patient, renal function was within normal limits, whereas it was abnormal in two patients. To our knowledge, this is the first report of cefepime induced asynchronous myoclonus and expressive aphasia in a patient with normal kidney function.

MitoTimer probe reveals the impact of autophagy, fusion, and motility on subcellular distribution of young and old mitochondrial protein and on relative mitochondrial protein age.

To study mitochondrial protein age dynamics, we targeted a time-sensitive fluorescent protein, MitoTimer, to the mitochondrial matrix. Mitochondrial age was revealed by the integrated portions of young (green) and old (red) MitoTimer protein. Mitochondrial protein age was dependent on turnover rates as pulsed synthesis, decreased import, or autophagic inhibition all increased the proportion of aged MitoTimer protein. Mitochondrial fusion promotes the distribution of young mitochondrial protein across the mitochondrial network as cells lacking essential fusion genes Mfn1 and Mfn2 displayed increased heterogeneity in mitochondrial protein age. Experiments in hippocampal neurons illustrate that the distribution of older and younger mitochondrial protein within the cell is determined by subcellular spatial organization and compartmentalization of mitochondria into neurites and soma. This effect was altered by overexpression of mitochondrial transport protein, RHOT1/MIRO1. Collectively our data show that distribution of young and old protein in the mitochondrial network is dependent on turnover, fusion, and transport.

Mitochondrial dynamics: the intersection of form and function.

Mitochondria within a cell exist as a population in a dynamic -morphological continuum. The balance of mitochondrial fusion and fission dictates a spectrum of shapes from interconnected networks to fragmented individual units. This plasticity bestows the adaptive flexibility needed to adjust to changing cellular stresses and metabolic demands. The mechanisms that regulate mitochondrial dynamics, their importance in normal cell biology, and the roles they play in disease conditions are only beginning to be understood. Dysfunction of mitochondrial dynamics has been identified as a possible disease mechanism in Parkinson's disease. This chapter will introduce the budding field of mitochondrial dynamics and explore unique characteristics of affected neurons in Parkinson's disease that increase susceptibility to disruptions in mitochondrial dynamics.

Redox proteomics analyses of the influence of co-expression of wild-type or mutated LRRK2 and Tau on C. elegans protein expression and oxidative modification: relevance to Parkinson disease.

AIMS: The human LRRK2 gene has been identified as the most common causative gene of autosomal-dominantly inherited and idiopathic Parkinson disease (PD). The G2019S substitution is the most common mutation in LRRK2. The R1441C mutation also occurs in cases of familial PD, but is not as prevalent. Some cases of LRRK2-based PD exhibit Tau pathology, which suggests that alterations on LRRK2 activity affect the pathophysiology of Tau. To investigate how LRRK2 might affect Tau and the pathophysiology of PD, we generated lines of C. elegans expressing human LRRK2 [wild-type (WT) or mutated (G2019S or R1441C)] with and without V337M Tau. Expression and redox proteomics were used to identify the effects of LRRK2 (WT and mutant) on protein expression and oxidative modifications. RESULTS: Co-expression of WT LRRK2 and Tau led to increased expression of numerous proteins, including several 60S ribosomal proteins, mitochondrial proteins, and the V-type proton ATPase, which is associated with autophagy. C. elegans expressing mutant LRRK2 showed similar changes, but also showed increased protein oxidation and lipid peroxidation, the latter indexed as increased protein-bound 4-hydroxy-2-nonenal (HNE). INNOVATION: Our study brings new knowledge about the possible alterations induced by LRRK2 (WT and mutated) and Tau interactions, suggesting the involvement of G2019S and R1441C in Tau-dependent neurodegenerative processes. CONCLUSION: These results suggest that changes in LRRK2 expression or activity lead to corresponding changes in mitochondrial function, autophagy, and protein translation. These findings are discussed with reference to the pathophysiology of PD.

Regulation of physiologic actions of LRRK2: focus on autophagy.

BACKGROUND: Mutations in LRRK2 are associated with familial and sporadic Parkinson's disease (PD). Subjects with PD caused by LRRK2 mutations show pleiotropic pathology that can involve inclusions containing α-synuclein, tau or neither protein. The mechanisms by which mutations in LRRK2 lead to this pleiotropic pathology remain unknown. OBJECTIVES: To investigate mechanisms by which LRRK2 might cause PD. METHODS: We used systems biology to investigate the transcriptomes from human brains, human blood cells and Caenorhabditis elegans expressing wild-type LRRK2. The role of autophagy was tested in lines of C. elegans expressing LRRK2, V337M tau or both proteins. Neuronal function was measured by quantifying thrashing. RESULTS: Genes regulating autophagy were coordinately regulated with LRRK2. C. elegans expressing V337M tau showed reduced thrashing, as has been noted previously. Coexpressing mutant LRRK2 (R1441C or G2019S) with V337M tau increased the motor deficits. Treating the lines of C. elegans with an mTOR inhibitor that enhances autophagic flux, ridaforolimus, increased the thrashing behavior to the same level as nontransgenic nematodes. CONCLUSION: These data support a role for LRRK2 in autophagy, raise the possibility that deficits in autophagy contribute to the pathophysiology of LRRK2, and point to a potential therapeutic approach addressing the pathophysiology of LRRK2 in PD.

Watching worms whither: modeling neurodegeneration in C. elegans.

Caenorhabditis elegans is increasingly being used to study neurodegenerative diseases. Nematodes are translucent, which facilitates study of particular neurons in the living animal, and easy to manipulate genetically. Despite vast evolutionary divergence, human proteins are functionally active when expressed in C. elegans, and disease-linked mutations in these proteins also cause phenotypic changes in the nematode. In this chapter, we review use of C. elegans to investigate the pathophysiology of Alzheimer's disease, Parkinson's disease, and axonal degeneration. Studies of presenilin, ß-amyloid, tau, α-synuclein, and LRRK2 all produce strong phenotypic effects in C. elegans, and in many cases reproduce selective neuronal vulnerability observed in humans. Disease-linked mutations enhance degeneration in the C. elegans models. These studies are increasingly leading to high-throughput screens that identify novel genes and novel pharmaceuticals that modify the disease course.

MKK6 binds and regulates expression of Parkinson's disease-related protein LRRK2.

Mutations in leucine-rich repeat kinase 2 (LRRK2) are prevalent causes of late-onset Parkinson's disease. Here, we show that LRRK2 binds to MAPK kinases (MKK) 3, 6, and 7, and that LRRK2 is able to phosphorylate MKK3, 6 and 7. Over-expression of LRRK2 and MKK6 increased the steady state levels of each protein beyond that observed with over-expression of either protein alone. Co-expression increased levels of MKK6 in the membrane more than in the cytoplasm. The increased expression of LRRK2 and MKK6 requires MKK6 activity. The disease-linked LRRK2 mutations, G2019S, R1441C and I2020T, enhance binding of LRRK2 to MKK6. This interaction was further supported by in vivo studies in C. elegans. RNAi knockdown in C. elegans of the endogenous orthologs for MKK6 or p38, sek-1 and pmk-1, abolishes LRRK2-mediated protection against mitochondrial stress. These results were confirmed by deletion of sek-1 in C. elegans. These data demonstrate that MKKs and LRRK2 function in similar biological pathways, and support a role for LRRK2 in modulating the cellular stress response.

LRRK2 modulates vulnerability to mitochondrial dysfunction in Caenorhabditis elegans.

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause autosomal-dominant familial Parkinson's disease. We generated lines of Caenorhabditis elegans expressing neuronally directed human LRRK2. Expressing human LRRK2 increased nematode survival in response to rotenone or paraquat, which are agents that cause mitochondrial dysfunction. Protection by G2019S, R1441C, or kinase-dead LRRK2 was less than protection by wild-type LRRK2. Knockdown of lrk-1, the endogenous ortholog of LRRK2 in C. elegans, reduced survival associated with mitochondrial dysfunction. C. elegans expressing LRRK2 showed rapid loss of dopaminergic markers (DAT::GFP fluorescence and dopamine levels) beginning in early adulthood. Loss of dopaminergic markers was greater for the G2019S LRRK2 line than for the wild-type line. Rotenone treatment induced a larger loss of dopamine markers in C. elegans expressing G2019S LRRK2 than in C. elegans expressing wild-type LRRK2; however, loss of dopaminergic markers in the G2019S LRRK2 nematode lines was not statistically different from that in the control line. These data suggest that LRRK2 plays an important role in modulating the response to mitochondrial inhibition and raises the possibility that mutations in LRRK2 selectively enhance the vulnerability of dopaminergic neurons to a stressor associated with Parkinson's disease.

Investigating convergent actions of genes linked to familial Parkinson's disease.

BACKGROUND: Mutations in LRRK2 are among the most frequent genetic changes identified in Parkinson's disease (PD), but how LRRK2 contributes to the pathophysiology of PD is not known. OBJECTIVES: To investigate how expressing wild-type or G2019S LRRK2 modifies cellular responses to rotenone, a mitochondrial toxin. METHODS: We investigated the vulnerability to mitochondrial toxins in Caenorhabditis elegans expressing wild-type or G2019S LRRK2. RESULTS: We observed a powerful role for LRRK2 in mitochondrial biology. Overexpressing LRRK2 strongly protects C. elegans against rotenone toxicity. The G2019S LRRK2 construct also protected LRRK2 against rotenone, but to a lesser degree than wild-type LRRK2. Knockdown of lrk-1 potentiated rotenone toxicity. CONCLUSIONS: These data suggest that LRRK1/2 regulate mitochondrial physiology.

Selection of embryonic stem cell-derived enhanced green fluorescent protein-positive dopamine neurons using the tyrosine hydroxylase promoter is confounded by reporter gene expression in immature cell populations.

Transplantation of mouse embryonic stem (mES) cells can restore function in Parkinson disease models, but can generate teratomas. Purification of dopamine neurons derived from embryonic stem cells by fluorescence-activated cell sorting (FACS) could provide a functional cell population for transplantation while eliminating the risk of teratoma formation. Here we used the tyrosine hydroxylase (TH) promoter to drive enhanced green fluorescent protein (eGFP) expression in mES cells. First, we evaluated 2.5-kilobase (kb) and 9-kb TH promoter fragments and showed that clones generated using the 9-kb fragment produced significantly more eGFP+/TH+ neurons. We selected the 9-kb TH clone with the highest eGFP/TH overlap for further differentiation, FACS, and transplantation experiments. Grafts contained large numbers of eGFP+ dopamine neurons of an appropriate phenotype. However, there were also numerous eGFP+ cells that did not express TH and did not have a neuronal morphology. In addition, we found cells in the grafts representing all three germ layers. Based on these findings, we examined the expression of stem cell markers in our eGFP+ population. We found that a majority of eGFP+ cells were stage-specific embryonic antigen-positive (SSEA-1+) and that the genetically engineered clones contained more SSEA-1+ cells after differentiation than the original D3 mES cells. By negative selection of SSEA-1, we could isolate a neuronal eGFP+ population of high purity. These results illustrate the complexity of using genetic selection to purify mES cell-derived dopamine neurons and provide a comprehensive analysis of cell selection strategies based on tyrosine hydroxylase expression. Disclosure of potential conflicts of interest is found at the end of this article.

Stromal cell-derived inducing activity, Nurr1, and signaling molecules synergistically induce dopaminergic neurons from mouse embryonic stem cells.

To induce differentiation of embryonic stem cells (ESCs) into specialized cell types for therapeutic purposes, it may be desirable to combine genetic manipulation and appropriate differentiation signals. We studied the induction of dopaminergic (DA) neurons from mouse ESCs by overexpressing the transcription factor Nurr1 and coculturing with PA6 stromal cells. Nurr1-expressing ESCs (N2 and N5) differentiated into a higher number of neurons (approximately twofold) than the naïve ESCs (D3). In addition, N2/N5-derived cells contained a significantly higher proportion (>50%) of tyrosine hydroxylase (TH)+ neurons than D3 (<30%) and an even greater proportion of TH+ neurons (approximately 90%) when treated with the signaling molecules sonic hedgehog, fibroblast growth factor 8, and ascorbic acid. N2/N5-derived cells express much higher levels of DA markers (e.g., TH, dopamine transporter, aromatic amino acid decarboxylase, and G protein-regulated inwardly rectifying K+ channel 2) and produce and release a higher level of dopamine, compared with D3-derived cells. Furthermore, the majority of generated neurons exhibited electrophysiological properties characteristic of midbrain DA neurons. Finally, transplantation experiments showed efficient in vivo integration/generation of TH+ neurons after implantation into mouse striatum. Taken together, our results show that the combination of genetic manipulation(s) and in vitro cell differentiation conditions offers a reliable and effective induction of DA neurons from ESCs and may pave the way for future cell transplantation therapy in Parkinson's disease.

Selective COX-2 inhibition prevents progressive dopamine neuron degeneration in a rat model of Parkinson's disease.

Several lines of evidence point to a significant role of neuroinflammation in Parkinson's disease (PD) and other neurodegenerative disorders. In the present study we examined the protective effect of celecoxib, a selective inhibitor of the inducible form of cyclooxygenase (COX-2), on dopamine (DA) cell loss in a rat model of PD. We used the intrastriatal administration of 6-hydroxydopamine (6-OHDA) that induces a retrograde neuronal damage and death, which progresses over weeks. Animals were randomized to receive celecoxib (20 mg/kg/day) or vehicle starting 1 hour before the intrastriatal administration of 6-OHDA. Evaluation was performed in vivo using micro PET and selective radiotracers for DA terminals and microglia. Post mortem analysis included stereological quantification of tyrosine hydroxylase, astrocytes and microglia. 12 days after the 6-OHDA lesion there were no differences in DA cell or fiber loss between groups, although the microglial cell density and activation was markedly reduced in animals receiving celecoxib (p < 0.01). COX-2 inhibition did not reduce the typical astroglial response in the striatum at any stage. Between 12 and 21 days, there was a significant progression of DA cell loss in the vehicle group (from 40 to 65%) that was prevented by celecoxib. Therefore, inhibition of COX-2 by celecoxib appears to be able, either directly or through inhibition of microglia activation to prevent or slow down DA cell degeneration.

Cortico-hippocampal APP and NGF levels are dynamically altered by cholinergic muscarinic antagonist or M1 agonist treatment in normal mice.

To determine whether altered cholinergic neurotransmission can modify the long-term secretion of amyloid precursor protein (APP), endogenous levels of APP and nerve growth factor (NGF), we administered a selective M1 muscarinic receptor agonist (RS86) or the muscarinic antagonist, atropine, for 7 days in vivo into young adult mice (C57BL/6j). The levels of NGF and total APP in the hippocampus, frontal cortex, striatum, parietal cortex and cerebrospinal fluid (CSF) were examined by ELISA and Western blot. We found that this repeated i.m. administration of M1 receptor agonist resulted in decreased total APP levels in the hippocampus, frontal cortex and parietal cortex, and increased secreted alpha-APPs levels in the CSF. M1 agonist treatment also resulted in decreased NGF levels in the hippocampus and CSF. These effects of the M1 muscarinic agonist could be blocked by atropine, which by itself elevated tissue levels of total APP. Interestingly, we found that the decrease of total APP in the hippocampus and striatum after M1 agonist treatment inversely correlated with the change in NGF levels. These data suggest that a sustained increased cholinergic, M1-mediated neurotransmission will enhance secretion of alpha-APPs in CSF and adaptively reduce the levels of total APP and NGF in the corticohippocampal regions of normal mice. The dynamic and adaptive regulation linking total APP and NGF levels in normal adult mice is relevant for understanding the pathophysiology of conditions with cholinergic and APP related pathologies, like Alzheimer's disease and Down's syndrome.