Ellen Gerhardt

Functional role of brain-derived neurotrophic factor in neuroprotective autoimmunity: therapeutic implications in a model of multiple sclerosis

Brain-derived neurotrophic factor plays a key role in neuronal and axonal survival. Brain-derived neurotrophic factor is expressed in the immune cells in lesions of experimental autoimmune encephalomyelitis and multiple sclerosis, thus potentially mediating neuroprotective effects. We investigated the functional role of brain-derived neurotrophic factor in myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. Mice deficient for brain-derived neurotrophic factor in immune cells displayed an attenuated immune response in the acute phase of experimental autoimmune encephalomyelitis, but progressive disability with enhanced axonal loss in the chronic phase of the disease. In mice deficient for central nervous system-derived brain-derived neurotrophic factor via glial fibrillary acidic protein-crescentin-mediated deletion, a more severe course of experimental autoimmune encephalomyelitis and an overall increased axonal loss was observed. In a lentiviral approach, injection of brain-derived neurotrophic factor-overexpressing T cells led to a less severe course of experimental autoimmune encephalomyelitis and direct axonal protection. Our data imply a functional role of brain-derived neurotrophic factor in autoimmune demyelination by mediating axon protection.

The mitochondrial chaperone protein TRAP1 mitigates α-Synuclein toxicity.

Overexpression or mutation of α-Synuclein is associated with protein aggregation and interferes with a number of cellular processes, including mitochondrial integrity and function. We used a whole-genome screen in the fruit fly Drosophila melanogaster to search for novel genetic modifiers of human [A53T]α-Synuclein–induced neurotoxicity. Decreased expression of the mitochondrial chaperone protein tumor necrosis factor receptor associated protein-1 (TRAP1) was found to enhance age-dependent loss of fly head dopamine (DA) and DA neuron number resulting from [A53T]α-Synuclein expression. In addition, decreased TRAP1 expression in [A53T]α-Synuclein–expressing flies resulted in enhanced loss of climbing ability and sensitivity to oxidative stress. Overexpression of human TRAP1 was able to rescue these phenotypes. Similarly, human TRAP1 overexpression in rat primary cortical neurons rescued [A53T]α-Synuclein–induced sensitivity to rotenone treatment. In human (non)neuronal cell lines, small interfering RNA directed against TRAP1 enhanced [A53T]α-Synuclein–induced sensitivity to oxidative stress treatment. [A53T]α-Synuclein directly interfered with mitochondrial function, as its expression reduced Complex I activity in HEK293 cells. These effects were blocked by TRAP1 overexpression. Moreover, TRAP1 was able to prevent alteration in mitochondrial morphology caused by [A53T]α-Synuclein overexpression in human SH-SY5Y cells. These results indicate that [A53T]α-Synuclein toxicity is intimately connected to mitochondrial dysfunction and that toxicity reduction in fly and rat primary neurons and human cell lines can be achieved using overexpression of the mitochondrial chaperone TRAP1. Interestingly, TRAP1 has previously been shown to be phosphorylated by the serine/threonine kinase PINK1, thus providing a potential link of PINK1 via TRAP1 to α-Synuclein.

Cascade of caspase activation in potassium-deprived cerebellar granule neurons: targets for treatment with peptide and protein inhibitors of apoptosis.

Cerebellar granule neurons (CGN) cultured in the presence of serum and depolarizing potassium concentrations undergo apoptosis when switched to serum-free medium containing physiological potassium concentrations. Here we show that processing of the key protease, caspase-3, depends on the activation of caspase-9, but not of caspase-8. Selective peptide inhibitors of caspase-9 block processing of caspase-3 and caspase-8 and inhibit apoptosis, whereas a selective inhibitor of caspase-8 blocks neither processing of caspase-3 nor cell death. The data obtained with peptide inhibitors were confirmed by adenovirally mediated ectopic expression of the cytokine response modifier A (crmA), the baculovirus protein p35, and the X chromosome-linked inhibitor of apoptosis (XIAP). Further, caspase-8-activating death receptors do not mediate apoptosis in CGN and potassium withdrawal-induced apoptosis evolves unaltered in gld or lpr mice, which harbor mutations in the CD95/CD95 ligand system. Thus, neuronal apoptosis triggered by potassium deprivation is death receptor-independent but involves the mitochondrial pathway of caspase activation.

FasL (CD95L/APO-1L) Resistance of Neurons Mediated by Phosphatidylinositol 3-Kinase-Akt/Protein Kinase B-Dependent Expression of Lifeguard/Neuronal Membrane Protein 35

The contribution of Fas (CD95/APO-1) to cell death mechanisms of differentiated neurons is controversially discussed. Rat cerebellar granule neurons (CGNs) express high levels of Fas in vitro but are resistant to FasL (CD95L/APO-1L/CD178)-induced apoptosis. We here show that this resistance was mediated by a phosphatidylinositol 3-kinase (PI 3-kinase)-Akt/protein kinase B (PKB)-dependent expression of lifeguard (LFG)/neuronal membrane protein 35. Reduction of endogenous LFG expression by antisense oligonucleotides or small interfering RNA lead to increased sensitivity of CGNs to FasL-induced cell death and caspase-8 cleavage. The inhibition of PI 3-kinase activity sensitized CGNs to FasL-induced caspase-8 and caspase-3 processing and caspase-dependent fodrin cleavage. Pharmacological inhibition of PI 3-kinase, overexpression of the inhibitory protein IκB, or cotransfection of an LFG reporter plasmid with dominant-negative Akt/PKB inhibited LFG reporter activity, whereas overexpression of constitutively active Akt/PKB increased LFG reporter activity. Overexpression of LFG in CGNs interfered with the sensitization to FasL by PI 3-kinase inhibitors. In contrast to CGNs, 12 glioma cell lines, which are sensitive to FasL, did not express LFG. Gene transfer of LFG into these FasL-susceptible glioma cells protected against FasL-induced apoptosis. These results demonstrate that LFG mediated the FasL resistance of CGNs and that, under certain circumstances, e.g., inhibition of the PI 3-kinase-Akt/PKB pathway, CGNs were sensitized to FasL.

Systematic Comparison of the Effects of Alpha-synuclein Mutations on Its Oligomerization and Aggregation

Aggregation of alpha-synuclein (ASYN) in Lewy bodies and Lewy neurites is the typical pathological hallmark of Parkinsons disease (PD) and other synucleinopathies. Furthermore, mutations in the gene encoding for ASYN are associated with familial and sporadic forms of PD, suggesting this protein plays a central role in the disease. However, the precise contribution of ASYN to neuronal dysfunction and death is unclear. There is intense debate about the nature of the toxic species of ASYN and little is known about the molecular determinants of oligomerization and aggregation of ASYN in the cell. In order to clarify the effects of different mutations on the propensity of ASYN to oligomerize and aggregate, we assembled a panel of 19 ASYN variants and compared their behaviour. We found that familial mutants linked to PD (A30P, E46K, H50Q, G51D and A53T) exhibited identical propensities to oligomerize in living cells, but had distinct abilities to form inclusions. While the A30P mutant reduced the percentage of cells with inclusions, the E46K mutant had the opposite effect. Interestingly, artificial proline mutants designed to interfere with the helical structure of the N-terminal domain, showed increased propensity to form oligomeric species rather than inclusions. Moreover, lysine substitution mutants increased oligomerization and altered the pattern of aggregation. Altogether, our data shed light into the molecular effects of ASYN mutations in a cellular context, and established a common ground for the study of genetic and pharmacological modulators of the aggregation process, opening new perspectives for therapeutic intervention in PD and other synucleinopathies.

Chemotherapy-induced cell death in primary cerebellar granule neurons but not in astrocytes: in vitro paradigm of differential neurotoxicity.

The nervous system is frequently the site of symptomatic toxicity of antineoplastic agents. However, there is limited information about the differential vulnerability of neurons, astrocytes and glioma cells. We have analyzed the effects of four chemotherapeutic drugs (lomustine, cisplatin, topotecan and vincristine) on primary cerebellar granule neurons and astrocytes derived from rats. All drugs led to cell death in cerebellar granule neurons in a concentration‐dependent manner. Comparison of the EC50 values for cerebellar neurons and astrocytes with the median EC50 values of 12 malignant glioma cell lines demonstrated a large therapeutic range for lomustin and cisplatin. Further, this comparison revealed a 100‐fold higher sensitivity of cerebellar neurons towards vincristine and 10‐fold higher sensitivity towards topotecan compared with glioma cells. Astrocytes were generally resistant to vincristine. In cerebellar granule neurons, vincristine and to a lesser extent topotecan induced caspase 3 and caspase 9 cleavage, and enhanced caspase activity and Akt‐dependent expression of phosphorylated BAD. zVAD‐fmk, a caspase inhibitor and brain‐derived neurotrophic factor (BDNF), but not MK‐801, a non‐competitive NMDA receptor antagonist, significantly reduced vincristine‐ or topotecan‐induced cell death.

Elevated α-synuclein caused by SNCA gene triplication impairs neuronal differentiation and maturation in Parkinson's patient-derived induced pluripotent stem cells.

We have assessed the impact of α-synuclein overexpression on the differentiation potential and phenotypic signatures of two neural-committed induced pluripotent stem cell lines derived from a Parkinsons disease patient with a triplication of the human SNCA genomic locus. In parallel, comparative studies were performed on two control lines derived from healthy individuals and lines generated from the patient iPS-derived neuroprogenitor lines infected with a lentivirus incorporating a small hairpin RNA to knock down the SNCA mRNA. The SNCA triplication lines exhibited a reduced capacity to differentiate into dopaminergic or GABAergic neurons and decreased neurite outgrowth and lower neuronal activity compared with control cultures. This delayed maturation phenotype was confirmed by gene expression profiling, which revealed a significant reduction in mRNA for genes implicated in neuronal differentiation such as delta-like homolog 1 (DLK1), gamma-aminobutyric acid type B receptor subunit 2 (GABABR2), nuclear receptor related 1 protein (NURR1), G-protein-regulated inward-rectifier potassium channel 2 (GIRK-2) and tyrosine hydroxylase (TH). The differentiated patient cells also demonstrated increased autophagic flux when stressed with chloroquine. We conclude that a two-fold overexpression of α-synuclein caused by a triplication of the SNCA gene is sufficient to impair the differentiation of neuronal progenitor cells, a finding with implications for adult neurogenesis and Parkinson’s disease progression, particularly in the context of bioenergetic dysfunction.

Fas/CD95 Regulatory Protein Faim2 Is Neuroprotective after Transient Brain Ischemia

Death receptor (DR) signaling has a major impact on the outcome of numerous neurological diseases, including ischemic stroke. DRs mediate not only cell death signals, but also proinflammatory responses and cell proliferation. Identification of regulatory proteins that control the switch between apoptotic and alternative DR signaling opens new therapeutic opportunities. Fas apoptotic inhibitory molecule 2 (Faim2) is an evolutionary conserved, neuron-specific inhibitor of Fas/CD95-mediated apoptosis. To investigate its role during development and in disease models, we generated Faim2-deficient mice. The ubiquitous null mutation displayed a viable and fertile phenotype without overt deficiencies. However, lack of Faim2 caused an increase in susceptibility to combined oxygen–glucose deprivation in primary neurons in vitro as well as in caspase-associated cell death, stroke volume, and neurological impairment after cerebral ischemia in vivo. These processes were rescued by lentiviral Faim2 gene transfer. In summary, we provide evidence that Faim2 is a novel neuroprotective molecule in the context of cerebral ischemia.

Tau deletion promotes brain insulin resistance

The molecular pathways underlying tau pathology–induced synaptic/cognitive deficits and neurodegeneration are poorly understood. One prevalent hypothesis is that hyperphosphorylation, misfolding, and fibrillization of tau impair synaptic plasticity and cause degeneration. However, tau pathology may also result in the loss of specific physiological tau functions, which are largely unknown but could contribute to neuronal dysfunction. In the present study, we uncovered a novel function of tau in its ability to regulate brain insulin signaling. We found that tau deletion leads to an impaired hippocampal response to insulin, caused by altered IRS-1 and PTEN (phosphatase and tensin homologue on chromosome 10) activities. Our data also demonstrate that tau knockout mice exhibit an impaired hypothalamic anorexigenic effect of insulin that is associated with energy metabolism alterations. Consistently, we found that tau haplotypes are associated with glycemic traits in humans. The present data have far-reaching clinical implications and raise the hypothesis that pathophysiological tau loss-of-function favors brain insulin resistance, which is instrumental for cognitive and metabolic impairments in Alzheimer’s disease patients.

Aggregate formation and toxicity by wild‐type and R621C synphilin‐1 in the nigrostriatal system of mice using adenoviral vectors

Synphilin‐1 was described as a protein interacting with α‐synuclein and is commonly found in Lewy bodies, the pathological hallmark of Parkinson’s disease (PD). Our group has previously described and characterized in vitro a mutation in the synphilin‐1 gene (R621C) in PD patients. Providing the first characterization of synphilin‐1 expression in an animal model, we here used adenoviral gene transfer to study the effects of wild‐type (WT) and R621C synphilin‐1 in dopaminergic neurons in mouse brain. As synphilin‐1 is commonly used to trigger aggregation of α‐synuclein in cell culture, we investigated not only non‐transgenic C57Bl/6 mice but also A30P‐α‐synuclein transgenic animals. Both WT synphilin‐1 and R621C synphilin‐1 led to the formation of Thioflavine‐S positive inclusions in C57Bl/6 mice and degeneration of dopaminergic neurons in the substantia nigra. R621C synphilin‐1 induced more aggregate formation than WT synphilin‐1 in A30P‐α‐synuclein transgenic mice, consistent with the role of the R621C mutation as a susceptibility factor for PD. Synphilin‐1 expression may be used to improve current mouse models of PD, as it induced both the formation of aggregates and degeneration of dopaminergic neurons, two core characteristics of PD that have not been well reproduced with expression of α‐synuclein.