Manfred Windisch

Glutaminyl cyclase inhibition attenuates pyroglutamate Aβ and Alzheimer's disease–like pathology

Because of their abundance, resistance to proteolysis, rapid aggregation and neurotoxicity, N-terminally truncated and, in particular, pyroglutamate (pE)-modified Aβ peptides have been suggested as being important in the initiation of pathological cascades resulting in the development of Alzheimers disease. We found that the N-terminal pE-formation is catalyzed by glutaminyl cyclase in vivo. Glutaminyl cyclase expression was upregulated in the cortices of individuals with Alzheimers disease and correlated with the appearance of pE-modified Aβ. Oral application of a glutaminyl cyclase inhibitor resulted in reduced Aβ3(pE)–42 burden in two different transgenic mouse models of Alzheimers disease and in a new Drosophila model. Treatment of mice was accompanied by reductions in Aβx–40/42, diminished plaque formation and gliosis and improved performance in context memory and spatial learning tests. These observations are consistent with the hypothesis that Aβ3(pE)–42 acts as a seed for Aβ aggregation by self-aggregation and co-aggregation with Aβ1–40/42. Therefore, Aβ3(pE)–40/42 peptides seem to represent Aβ forms with exceptional potency for disturbing neuronal function. The reduction of brain pE-Aβ by inhibition of glutaminyl cyclase offers a new therapeutic option for the treatment of Alzheimers disease and provides implications for other amyloidoses, such as familial Danish dementia.

Sodium selenate specifically activates PP2A phosphatase, dephosphorylates tau and reverses memory deficits in an Alzheimer's disease model.

Neurofibrillary tangles composed of abnormally hyperphosphorylated tau protein are a hallmark of Alzheimers disease (AD) and related tauopathies. Tau hyperphosphorylation is thought to promote aggregation with subsequent tangle formation. Reducing tau phosphorylation by boosting the activity of the key phosphatase/s that mediate dephosphorylation of tau could be a viable clinical strategy in AD. One of the key phosphatases implicated in regulating tau protein phosphorylation is the serine-threonine phosphatase PP2A. We have determined that sodium selenate can act as a specific agonist for PP2A, significantly boosting phosphatase activity. Acute treatment of either neuroblastoma cells or normal aged mice with sodium selenate rapidly reduced tau protein phosphorylation. Sodium selenate-treated transgenic TAU441 mice had significantly lower levels of phospho- and total tau levels in the hippocampus and amygdala compared with controls and exhibited significantly improved spatial learning and memory on the Morris Water Maze task. Sodium selenate is a specific activator of PP2A with excellent oral bioavailability, and favourable central nervous system penetrating properties. Clinical studies in patients with AD are envisaged in the near future.

Lysosomal pathology associated with α‐synuclein accumulation in transgenic models using an eGFP fusion protein

Disorders with Lewy body (LB) formation, such as Parkinsons disease (PD) and dementia with Lewy bodies (DLB), are characterized by α‐synuclein accumulation in the neuronal cell body. Recent studies have suggested that in addition to LBs, α‐synuclein might accumulate more widely throughout the neurons and their processes, leading to neurodegeneration and functional impairment. The precise patterns of α‐synuclein accumulation in vivo, however, and its relationship with subcellular neuronal alterations such as lysosomal pathology are not completely clear. To this end, we developed transgenic (tg) in vivo and in vitro models expressing a stable enhanced green fluorescent protein (eGFP) tagged in the C‐terminal site of a human (h)α‐synuclein construct under the regulatory control of the platelet‐derived growth factor‐β (PDGFβ) promoter and carried out confocal, ultrastructural, and biochemical studies. In tg mice, confocal studies demonstrated a wide distribution of hα‐synuclein–eGFP in the neuronal cell bodies, axons, and presynaptic terminals. In several neuronal cell bodies and their neurites, hα‐synuclein–eGFP was found not only as inclusions but also as discrete granular structures that in double‐labeling studies colocalized with antibodies against hα‐synuclein and the lysosomal marker cathepsin D. Consistent with these findings, ultrastructural analysis showed that hα‐synuclein–eGFP overexpression resulted in the accumulation of electrodense inclusions and laminated bodies suggestive of lysosomal pathology, and that the hα‐synuclein–eGFP protein was more abundant in the lysosomal fractions of the tg animals. Taken together, these findings support the notion that enhanced visualization of α‐synuclein utilizing a hybrid eGFP molecule reveals a more widespread accumulation of this molecule in several neuronal compartments, promoting lysosomal dysfunction. Furthermore, the PDGFβ–hα‐synuclein–eGFP tg model might be a valuable tool in testing new treatments for LBD in a fast and reliable manner.

The neuroprotective effects of Cerebrolysin in a transgenic model of Alzheimer's disease are associated with improved behavioral performance.

Summary. Cerebrolysin™ is a peptide mixture with neurotrophic effects that might have the ability of both reducing amyloid burden and improving synaptic plasticity in Alzheimer’s disease (AD). In order to determine if Cerebrolysin is capable of ameliorating the neurodegenerative and behavioral alterations associated with amyloid β (Aβ) production; transgenic (tg) mice expressing mutant human amyloid precursor protein (APP) under the Thy1 promoter were treated with Cerebrolysin or saline alone starting at 3 or 6 months of age for a total of three months. Animals were then tested behaviorally (at 6 and 9 months of age respectively) in the water maze and then analyzed neuropathologically for amyloid burden, synaptic density, astrogliosis and apoptosis. Performance analysis in the water maze showed that in the younger tg mice cohort, Cerebrolysin treatment significantly ameliorated the performance deficits. In the older cohort, there was a trend toward improved performance in the learning curve. Neuropathological examination showed that in both age/treatment groups, Cerebrolysin promoted synaptic regeneration, and reduced the proportion of neurons displaying DNA fragmentation by the (TdT)-mediated dUTP-biotin nick-end labeling (TUNEL) method. Moreover, Cerebrolysin treatment reduced Aβ burden by 43% in the young group and by 27% in the older group. Taken together, these results suggest that Cerebrolysin treatment might have beneficial effects in patients with cognitive impairment by reducing Aβ accumulation and promoting the preservation of synaptic terminals.

Alzheimer's Therapeutics Targeting Amyloid Beta 1–42 Oligomers I: Abeta 42 Oligomer Binding to Specific Neuronal Receptors Is Displaced by Drug Candidates That Improve Cognitive Deficits

Synaptic dysfunction and loss caused by age-dependent accumulation of synaptotoxic beta amyloid (Abeta) 1–42 oligomers is proposed to underlie cognitive decline in Alzheimers disease (AD). Alterations in membrane trafficking induced by Abeta oligomers mediates reduction in neuronal surface receptor expression that is the basis for inhibition of electrophysiological measures of synaptic plasticity and thus learning and memory. We have utilized phenotypic screens in mature, in vitro cultures of rat brain cells to identify small molecules which block or prevent the binding and effects of Abeta oligomers. Synthetic Abeta oligomers bind saturably to a single site on neuronal synapses and induce deficits in membrane trafficking in neuronal cultures with an EC50 that corresponds to its binding affinity. The therapeutic lead compounds we have found are pharmacological antagonists of Abeta oligomers, reducing the binding of Abeta oligomers to neurons in vitro, preventing spine loss in neurons and preventing and treating oligomer-induced deficits in membrane trafficking. These molecules are highly brain penetrant and prevent and restore cognitive deficits in mouse models of Alzheimers disease. Counter-screening these compounds against a broad panel of potential CNS targets revealed they are highly potent and specific ligands of the sigma-2/PGRMC1 receptor. Brain concentrations of the compounds corresponding to greater than 80% receptor occupancy at the sigma-2/PGRMC1 receptor restore cognitive function in transgenic hAPP Swe/Ldn mice. These studies demonstrate that synthetic and human-derived Abeta oligomers act as pharmacologically-behaved ligands at neuronal receptors - i.e. they exhibit saturable binding to a target, they exert a functional effect related to their binding and their displacement by small molecule antagonists blocks their functional effect. The first-in-class small molecule receptor antagonists described here restore memory to normal in multiple AD models and sustain improvement long-term, representing a novel mechanism of action for disease-modifying Alzheimers therapeutics.

Neurotrophic activities and therapeutic experience with a brain derived peptide preparation

In spite that the use of naturally occurring neurotrophic factors like NGF, BDNF, CNTF, GDNF and others for treatment of neurodegenerative disorders seems promising because of their pharmacological properties, until now no large scale clinical trials have been published. One of the reasons is that these molecules are unable to penetrate through the blood brain barrier, making invasive application strategies like intracerebroventricular infusion necessary. Another one is the fact that in first clinical studies, several undesirable side-effects like hyperalgesia or weight loss have been reported. Major efforts are now put into development of improved application procedures and in treatment protocols for avoiding the known side-effects. Already 7 years ago it has been demonstrated that Cerebrolysin, a peptidergic drug, produced from purified brain proteins by standardized enzymatic breakdown, containing biologically active peptides, is exerting nerve growth factor like activity on neurons from dorsal root ganglia. Still ongoing investigations are showing growth promoting efficacy of this drug in different neuronal populations from peripheral and central nervous system. The current findings are in accordance with several older publications, enabling now a more clear interpretation of these findings. In addition to the direct neurotrophic effect, the drug also shows clear neuroprotective properties after different types of lesion in vitro and in vivo, resembling the pharmacological activities of naturally occurring nerve growth factors. Neurotrophic and neuroprotective efficacy has been shown with a broad variety of methods in different models and it is remarkable that all biochemical and morphological drug dependent alterations are resulting in improvements of learning and memory. Because of these experimental results, clinical trials using cerebrolysin in Alzheimers patients have been performed, demonstrating a quick improvement in the overall state of the patients, particularly enhancing the cognitive performance. It is remarkable that these effects are long lasting after cessation of the active treatment procedure. Even 6 months after stop of drug application improvements in AD-patients are detectable. Therefore it is concluded that cerebrolysin is able to induce repair phenomena, resulting in long term stabilization. In contrast to the naturally occurring growth factors, tolerability of this drug is extremely high, without any reports about serious side-effects in these clinical studies.

Antiapoptotic effects of the peptidergic drug Cerebrolysin on primary cultures of embryonic chick cortical neurons

Summary. Cerebrolysin (EBEWE Arzneimittel, Austria, Europe) is a widely used drug relieving the symptoms of a variety of neurological disorders, particularly of neurodegenerative dementia of the Alzheimers type. It consists of approximately 25% of low molecular weight peptides (<10 k DA) and a mixture of approximately 75% free amino acids, this being based on the total nitrogen content.In this study we used a low serum (2% serum supplement) cell stress in-vitro model to assess drug effectiveness on neuronal viability and programmed cell death (PCD). In this in-vitro model the type of cell death was previously shown to be primarly apoptotic, which was verified by DNA-laddering and TUNEL-staining. For evaluation of neuronal viability a MTT-reduction assay was performed after 4 DIV and 8 DIV and the percentage of apoptotic neurons was determined by bis-benzimide staining of nuclear chromatin.To differentiate between possible effects of the free amino acids and the peptide fraction of Cerebrolysin an artificial amino acid mixture (AA-mix) was used as a control.Cerebrolysin, the AA-mix and 10% foetal calf serum (FCS) caused a similar increase in viability after 4 DIV, whereas the effects of the growth factors BDNF and FGF-2 were less pronounced. After 8 DIV Cerebrolysin, but not the AA-mix, was able to ameliorate neuronal viability, which could reflect a neuro-protective effect or an increased activity of the mitochondrial dehydrogenase measured in a MTT-reduction assay. The percentage of cells showing apoptotic chromatin changes was significantly reduced (p < 0.01) in cultures treated with Cerebrolysin, whereas the AA-mix failed to decrease the percentage of cells showing apoptotic chromatin changes. These findings ascertain an anti-apoptotic effect of the peptide fraction of Cerebrolysin and reveal a transient viability promoting effect of the amino acid fraction, which is most likely due to improved nutritional supply.

Effects of Cerebrolysin on amyloid-beta deposition in a transgenic model of Alzheimer's disease.

We investigated the potential mechanisms through which Cerebrolysin, a neuroprotective noothropic agent, might affect Alzheimers disease pathology. Transgenic (tg) mice expressing mutant human (h) amyloid precursor protein 751 (APP751) cDNA under the Thy-1 promoter (mThy1-hAPP751) were treated for four weeks with this compound and analyzed by confocal microscopy to asses its effects on amyloid plaque formation and neurodegeneration. In this model, amyloid plaques in the brain are found much earlier (beginning at 3 months) than in other tg models. Quantitative computer-aided analysis with anti-amyloid-beta protein (A beta) antibodies, revealed that Cerebrolysin significantly reduced the amyloid burden in the frontal cortex of 5-month-old mice. Furthermore, Cerebrolysin treatment reduced the levels of A beta(1-42). This was accompanied by amelioration of the synaptic alterations in the frontal cortex of mThy1-hAPP751 tg mice. In conclusion, the present study supports the possibility that Cerebrolysin might have neuroprotective effects by decreasing the production of A beta(1-42) and reducing amyloid deposition.

Development of a new treatment for alzheimer’s disease and parkinson’s disease using anti-aggregatory β-synuclein-derived peptides

The synaptic protein α-synuclein is a major constitutent of Lewy bodies (LB), pathological neuronal inclusion bodies found in Parkinson’s disease (PD), Alzheimer’s disease (AD), and other neurodegenerative disorders. Owing to data from patient brains, it was speculated that an imbalance between α-synuclein and β-synuclein might be one of the reasons for formation of LBs and the consequent functional deficits. This was supported by the fact that β-synuclein is able to prevent abnormal α-synuclein aggregation.Transgenic mice overexpressing α-synuclein display LB-like inclusions in different brain regions and motor deficits. To verify if re-establishing a normal relation between α-synuclein and β-synuclein is able to prevent the pathology, bigenic mice have been created that overexpress both synucleins. β-synuclein decreased formation of LBs by 40% and prevented functional deficits. This is considered as preliminary in vivo proof of anti-aggregatory function of β-synuclein and its potential as therapeutic substance for treatment of neurodegenerative disorders linked with abnormal protein aggregation.Peptide libraries have been synthesized to explore the active structures of β-synuclein. The first 15 N-terminal amino-acids turned out to be important for the antiaggregatory effect. Further smaller β-synuclein-derived peptides have screened for antiaggregatory and neuroprotective potency in different tissue-culture systems. Preliminary data suggest some of them can be used as leads for further drug development.

Cerebrolysin in Alzheimer's disease: a randomized, double-blind, placebo-controlled trial with a neurotrophic agent

Summary.Summary. Cerebrolysin (Cere) is a compound with neurotrophic activity. It has been shown to be effective in the treatment of Alzheimers disease (AD) in earlier trials. In this multicenter, randomized, double-blind, placebo-controlled, parallel-group study, patients were injected intravenously with placebo or 30 mL Cere five days per week for four weeks. Effects on cognition and global function were evaluated with the Alzheimer Disease Assessment Scale – Cognitive Subscale (ADAS-Cog) and the Clinicians Interview-based Impression of Change with Caregiver Input scale (CIBIC+) 4, 12, 24 weeks after the beginning of the injections. 192 patients were enrolled, 95 were randomized to placebo, and 97 to Cere. At baseline, there was a significant difference between groups for age, age of onset of dementia, and the number of patients with hallucinations. At week 12 there was a significant difference on the CIBIC+ (p = 0.033) in favor of Cere. The number of CIBIC+ responders (score ≤ 4), was significantly higher (p = 0.007), with 68 (76%) in the Cere group and 51 (57%) in the placebo group. Trends were noted in the Disability Assessment in Dementia scale and the Cornell Depression Scale. Adverse events were recorded in 73% of placebo and 64% of Cere patients. Most common adverse events were headaches, dizziness, weight loss and anxiety.Conclusions: Cere treatment was well tolerated and resulted in significant improvements in the global score two months after the end of active treatment.