Peter Schmid

Transient production of TGF-beta 2 by postnatal cerebellar neurons and its effect on neuroblast proliferation

The beta transforming growth factors (TGF‐β) are suggested to regulate developmental processes since they are distinctly expressed during embryogenesis and exert pleiotropic effects on cell growth and differentiation. In the present study the expression of TGF‐β isoforms was investigated in the postnatal and adult mouse brain. As shown by in situ hybridization, TGF‐β2 was expressed in the choroid plexus, hippocampus, dentate gyrus and cerebellar Purkinje neurons, both postnatally and in adults. Furthermore, TGF‐β2 expression was observed postnatally in immature cerebellar neurons of both the external and internal granule cell layers. In the external granule cell layer, the frequency of TGF‐β2 transcripts increased until postnatal day 10 and declined thereafter. In contrast to TGF‐β2, no TGF‐β1 mRNA was detected in cerebellar granule cells. TGF‐β3 expression was widely distributed in postnatal brains although at very low levels. The significance of TGF‐β2 production by cerebellar granule cells was further investigated using cultures of small cerebellar neurons. In these cultures reverse polymerase chain reaction analysis revealed expression of TGF‐β2 but low or almost undetectable levels of TGF‐β1 or ‐β3 mRNAs. Likewise, only TGF‐β2 protein in its latent form was identified in the culture supernatant; the release of TGF‐β2 was maximal during the second day in vitro. Furthermore, TGF‐β was found to inhibit the proliferation of cultured small cerebellar neurons. Taken together, these data suggest that TGF‐β2 is involved in the regulation of postnatal development of the cerebellum.

Development of Highly Organized Lymphoid Structures in Buruli Ulcer Lesions after Treatment with Rifampicin and Streptomycin

Background Buruli ulcer caused by Mycobacterium ulcerans is an infection of the subcutaneous tissue leading to chronic necrotising skin ulcers. The pathogenesis is associated with the cytocidal and immunosuppressive activities of a macrolide toxin. Histopathological hallmark of progressing disease is a poor inflammatory response despite of clusters of extracellular bacilli. While traditionally wide excision of the infected tissue was the standard treatment, provisional WHO guidelines now recommend an eight week pre-treatment with streptomycin and rifampicin. Methodology/Principal Findings We conducted a detailed immunohistochemical analysis of tissue samples from Buruli patients who received antibiotic treatment. Cellular immune response along with bacterial load and distribution were monitored. We demonstrate that this treatment leads to the development of highly organized cellular infiltration surrounding areas of coagulative necrosis. Diffuse infiltrates, granulomas and dense lymphocyte aggregation close to vessels were observed. Mycobacterial material was primarily located inside mononuclear phagocytes and microcolonies consisting of extracellular rod-shaped mycobacteria were no longer found. In observational studies some patients showed no clinical response to antibiotic treatment. Corresponding to that, one of five lesions analysed presented with huge clusters of rod-shaped bacilli but no signs of infiltration. Conclusions/Significance Results signify that eight weeks of antibiotic treatment reverses local immunosuppression and leads to an active inflammatory process in different compartments of the skin. Structured leukocyte infiltrates with unique signatures indicative for healing processes developed at the margins of the lesions. It remains to be analysed whether antibiotic resistance of certain strains of M. ulcerans, lacking patient compliance or poor drug quality are responsible for the absent clinical responses in some patients. In future, analysis of local immune responses could serve as a suitable surrogate marker for the efficacy of alternative treatment strategies.

A novel BACE inhibitor NB-360 shows a superior pharmacological profile and robust reduction of amyloid-β and neuroinflammation in APP transgenic mice

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.

Localization of transforming growth factor-β1, -β2 and -β3 gene expression in bovine mammary gland

Abstract We have studied the expression of transforming growth factor (TGF)-β1, -β2, and -β3 in the non-lactating and lactating bovine mammary gland by in situ hybridization. All three isoforms were expressed in the lobuloalveolar framework of the non-lactating and lactating gland although marked differences were apparent in their spatial distribution. TGF-β1 was expressed predominantly by the epithelial cells of the lobules although expression was also observed in the intralobular stroma cells lining the epithelium. In contrast, TGF-β2 expression was only observed in the epithelial cells. TGF-β3 transcripts were expressed at the highest levels and were observed in almost all cells of the lobule. No TGF-β signals were found in the interlobular regions of the mammary gland. The possible regulatory functions of these molecules in development of the mammary gland and on differentiation processes in the neonate are discussed.

In situ hybridization analysis of cytokine, proto-oncogene and tumour suppressor gene expression in psoriasis.

The purpose of this study was to investigate and to compare, by in situ hybridization, gene expression of IL-1Β, IL-8, TGF-Β1, TGF-Β2, TGF-Β3, TGF-α, p53 and c-myc in lesions and in non-involved skin of patients with psoriasis. All lesional skin biopsies showed overexpression of IL-1Β, IL-8 and TGF-α mRNAs. IL-1Β hybridization signals were strong in a small number of cells localized predominantly in the dermal papillae and in the suprapapillary epidermis. Overexpression of TGF-α was observed in all suprabasal keratinocytes, whereas strongly elevated IL-8 mRNA expression was found to be restricted to clusters of suprabasal keratinocytes. TGF-Β3, p53 and c-myc transcripts were clearly detected in the epidermis of all biopsies, although expression levels were comparable in lesional and non-lesional skin.

Mutant Huntingtin Gene-Dose Impacts on Aggregate Deposition, DARPP32 Expression and Neuroinflammation in HdhQ150 Mice

Huntingtons disease (HD) is an autosomal dominant, progressive and fatal neurological disorder caused by an expansion of CAG repeats in exon-1 of the huntingtin gene. The encoded poly-glutamine stretch renders mutant huntingtin prone to aggregation. HdhQ150 mice genocopy a pathogenic repeat (∼150 CAGs) in the endogenous mouse huntingtin gene and model predominantly pre-manifest HD. Treating early is likely important to prevent or delay HD, and HdhQ150 mice may be useful to assess therapeutic strategies targeting pre-manifest HD. This requires appropriate markers and here we demonstrate, that pre-symptomatic HdhQ150 mice show several dramatic mutant huntingtin gene-dose dependent pathological changes including: (i) an increase of neuronal intra-nuclear inclusions (NIIs) in brain, (ii) an increase of extra-nuclear aggregates in dentate gyrus, (iii) a decrease of DARPP32 protein and (iv) an increase in glial markers of neuroinflammation, which curiously did not correlate with local neuronal mutant huntingtin inclusion-burden. HdhQ150 mice developed NIIs also in all retinal neuron cell-types, demonstrating that retinal NIIs are not specific to human exon-1 R6 HD mouse models. Taken together, the striking and robust mutant huntingtin gene-dose related changes in aggregate-load, DARPP32 levels and glial activation markers should greatly facilitate future testing of therapeutic strategies in the HdhQ150 HD mouse model.

Fragments of HdhQ150 Mutant Huntingtin Form a Soluble Oligomer Pool That Declines with Aggregate Deposition upon Aging

Cleavage of the full-length mutant huntingtin (mhtt) protein into smaller, soluble aggregation-prone mhtt fragments appears to be a key process in the neuropathophysiology of Huntington’s Disease (HD). Recent quantification studies using TR-FRET-based immunoassays showed decreasing levels of soluble mhtt correlating with an increased load of aggregated mhtt in the aging HdhQ150 mouse brain. To better characterize the nature of these changes at the level of native mhtt species, we developed a detection method that combines size exclusion chromatography (SEC) and time-resolved fluorescence resonance energy transfer (TR-FRET) that allowed us to resolve and define the formation, aggregation and temporal dynamics of native soluble mhtt species and insoluble aggregates in the brain of the HdhQ150 knock-in mouse. We found that mhtt fragments and not full-length mhtt form oligomers in the brains of one month-old mice long before disease phenotypes and mhtt aggregate histopathology occur. As the HdhQ150 mice age, brain levels of soluble full-length mhtt protein remain similar. In contrast, the soluble oligomeric pool of mhtt fragments slightly increases during the first two months before it declines between 3 and 8 months of age. This decline inversely correlates with the formation of insoluble mhtt aggregates. We also found that the pool-size of soluble mhtt oligomers is similar in age-matched heterozygous and homozygous HdhQ150 mouse brains whereas insoluble aggregate formation is greatly accelerated in the homozygous mutant brain. The capacity of the soluble mhtt oligomer pool therefore seems exhausted already in the heterozygous state and likely kept constant by changes in flux and, as a consequence, increased rate of insoluble aggregate formation. We demonstrate that our novel findings in mice translate to human HD brain but not HD patient fibroblasts.

THE PATTERN OF PROTHYMOSIN ALPHA GENE EXPRESSION COINCIDES WITH THAT OF MYC PROTO-ONCOGENES DURING MOUSE EMBRYOGENESIS

SummaryThe nuclear protein prothymosin α is thought to play a critical role in cellular proliferation. Transcription of the gene encoding prothymosin α has been shown to be activated by the proto-oncogene c-myc. Also, prothymosin α mRNA expression correlates with that of c-myc in human colon cancer. We compared the previously reported embryonic expression pattern of the proto-oncogene c-myc and the pattern of the prothymosin α gene byin situ hybridization. Prothymosin α is transcribed in all tissues expressing c-myc, including brown adipose tissue, salivary gland, thymus and liver. In addition, we show that the prothymosin α gene is active in tissues expressing specifically N-myc such as the neuronal anlage and hair follicles in skin. Therefore, during mouse foetal development the temporal, spatial and tissue-specific expression patterns of bothmyc protooncogenes coincide with the pattern of prothymosin α.

Transgenic expression of β1 antibody in brain neurons impairs age-dependent amyloid deposition in APP23 mice

Heterologous expression of the functional amyloid beta (Aβ) antibody β1 in the central nervous system was engineered to maximize antibody exposure in the brain and assess the effects on Aβ production and accumulation in these conditions. A single open reading frame encoding the heavy and light chains of β1 linked by the mouth and foot virus peptide 2A was expressed in brain neurons of transgenic mice. Two of the resulting BIN66 transgenic lines were crossed with APP23 mice, which develop severe central amyloidosis. Brain concentrations at steady-state 5 times greater than those found after peripheral β1 administration were obtained. Similar brain and plasma β1 concentrations indicated robust antibody efflux from the brain. In preplaque mice, β1 formed a complex with Aβ that caused a modest Aβ increase in brain and plasma. At 11 months of age, β1 expression reduced amyloid by 97% compared with age-matched APP23 mice. Interference of β1 with β-secretase cleavage of amyloid precursor protein was relatively small. Our data suggest that severely impaired amyloid formation was primarily mediated by a complex of β1 with soluble Aβ, which might have prevented Aβ aggregation or favored transport out of the brain.

THE BACE INHIBITOR NB-360 HAS EXCELLENT BRAIN PENETRATION AND EFFICACY ON AMYLOID-B LOAD IN ANIMAL MODELS

human APOE3 or APOE4) were treated with Bex, LG268 (a more selective RXR agonist), or vehicle control in 3 treatment paradigms: T1) 7-day oral gavage (5.75-6M); T2) 7-day hydrogel (5.75-6M); and T3) 30-day hydrogel (5-6M). Hydrogel provides a steady dosage of drug throughout the awake period of the mice. Brains were harvested, dissected, and homogenized by 3-step serial extraction.Results: In brain regions with lowAb levels at treatment, RXR agonists did not change soluble levels of Ab 42 and oAb in E3FAD or E4FAD mice. In brain regions with intermediate Ab levels, RXR agonist treatment induced an increase in soluble Ab 42 and oAb levels in E3FAD and E4FADmice. However, in the hippocampus of E4FADmice, with high Ab levels at treatment, RXR agonists induced a decrease in soluble Ab 42 and oAb levels and an increase in synaptic proteins. Importantly, total apoE levels were unaffected for all treatment groups, suggesting an alternate mechanism of action for RXR agonists. Our data further demonstrate that the beneficial effects of RXR agonists in E4FAD mice are mediated via: increased ABCA1 and ABCG1 expression, increased apoE4 association with lipoproteins, increased apoE/Ab complex levels, reduced oAb levels and enhanced synaptic viability. Conclusions: Collectively, our data demonstrate that RXR agonist efficacy is determined by the levels of Ab pathology at time of treatment, exhibiting no effect, or even an increase the levels of neurotoxic Ab in prevention paradigms where Ab levels are likely sub-pathological. However, in later stages of AD, RXR agonists may address the loss of function associated with APOE4 by increasing apoE4 lipidation and apoE4/Ab complex formation. Future studies are necessary to determine whether this pathway is relevant for APOE3 carriers with high Ab pathology, or if RXR agonists are an APOE4specific AD therapeutic.