Stefan Hettwer

Coincident pre- and postsynaptic activation induces dendritic filopodia via neurotrypsin-dependent agrin cleavage.

The synaptic serine protease neurotrypsin is essential for cognitive function, as its deficiency in humans results in severe mental retardation. Recently, we demonstrated the activity-dependent release of neurotrypsin from presynaptic terminals and proteolytical cleavage of agrin at the synapse. Here we show that the activity-dependent formation of dendritic filopodia is abolished in hippocampal neurons from neurotrypsin-deficient mice. Administration of the neurotrypsin-dependent 22 kDa fragment of agrin rescues the filopodial response. Detailed analyses indicated that presynaptic action potential firing is necessary for the release of neurotrypsin, whereas postsynaptic NMDA receptor activation is necessary for the neurotrypsin-dependent cleavage of agrin. This contingency characterizes the neurotrypsin-agrin system as a coincidence detector of pre- and postsynaptic activation. As the resulting dendritic filopodia are thought to represent precursors of synapses, the neurotrypsin-dependent cleavage of agrin at the synapse may be instrumental for a Hebbian organization and remodeling of synaptic circuits in the CNS.

Neurotrypsin cleaves agrin locally at the synapse

The synaptic serine protease neurotryp sin is considered to be essential for the establishment and maintenance of cognitive brain functions, because humans lacking functional neurotrypsin suffer from severe mental retardation. Neurotrypsin cleaves agrin at two homologous sites, liberating a 90‐kDa and a C‐terminal 22‐kDa fragment from the N‐terminal moi ety of agrin. Morphological analyses indicate that neu rotrypsin is contained in presynaptic terminals and externalized in association with synaptic activity, while agrin is localized to the extracellular space at or in the vicinity of the synapse. Here, we present a detailed biochemical analysis of neurotrypsin‐mediated agrin cleavage in the murine brain. In brain homogenates, we found that neurotrypsin exclusively cleaves glycanated variants of agrin. Studies with isolated synaptosomes obtained by subcellular fractionation from brains of wild‐type and neurotrypsin‐overexpressing mice re vealed that neurotrypsin‐dependent cleavage of agrin was concentrated at synapses, where the most heavily glycanated variants of agrin predominate. Because agrin has been shown to play an important role in the formation and the maintenance of excitatory synapses in the central nervous system, its local cleavage at the synapse implicates the neurotrypsin/agrin system in the regulation of adaptive reorganizations of the synaptic circuitry in the context of cognitive functions, such as learning and memory.— Stephan, A., Mateos, J. M., Kozlov, S. V., Cinelli, P., Kistler, A. D., Hettwer, S., Rulicke, T., Streit, P., Kunz, B., Sonderegger, P. Neu rotrypsin cleaves agrin locally at the synapse. FASEB J. 22, 1861–1873 (2008)

Specific cleavage of agrin by neurotrypsin, a synaptic protease linked to mental retardation

The synaptic serine protease neurotrypsin is thought to be important for adaptive synaptic processes required for cognitive functions, because humans deficient in neurotrypsin suffer from severe mental retardation. In the present study, we describe the biochemical characterization of neurotrypsin and its so far unique substrate agrin. In cell culture experiment as well as in neurotrypsin‐deficient mice, we showed that agrin cleavage depends on neurotrypsin and occurs at two conserved sites. Neurotrypsin and agrin were expressed recombinantly, purified, and assayed in vitro. A catalytic efficiency of 1.3 × 104 M−1 • s−1 was determined. Neurotrypsin activity was shown to depend on calcium with an optimal activity in the pH range of 7–8.5. Mutagenesis analysis of the amino acids flanking the scissile bonds showed that cleavage is highly specific due to the unique substrate recognition pocket of neurotrypsin at the active site. The C‐termi‐nal agrin fragment released after cleavage has recently been identified as an inactivating ligand of the Na+/ K+‐ATPase at CNS synapses, and its binding has been demonstrated to regulate presynaptic excitability. Therefore, dysregulation of agrin processing is a good candidate for a pathogenetic mechanism underlying mental retardation. In turn, these results may also shed light on mechanisms involved in cognitive functions.—Reif, R., Sales, S., Hettwer, S., Dreier, B., Gisler, C., Wolfel, J., Luscher, D., Zurlinden, A., Stephan, A., Ahmed, S., Baici, A., Ledermann, B., Kunz, B., Sonderegger, P. Specific cleavage of agrin by neurotrypsin, a synaptic protease linked to mental retardation. FASEB J. 21, 3468–3478 (2007)

Elevated levels of a C-terminal agrin fragment identifies a new subset of sarcopenia patients

Sarcopenia is a recently defined medical condition described as age-associated loss of skeletal muscle mass and function. Recently, a transgenic mouse model was described linking dispersal of the neuromuscular junction caused by elevated agrin degradation to the rapid onset of sarcopenia. These mice show a significant elevation of serum levels of a C-terminal agrin fragment (CAF) compared to wild-type littermates. A series of experiments was designed to ascertain the significance of elevated agrin degradation in the development of human sarcopenia. A quantitative Western blot method was devised to detect CAF in sera of humans. A first trial on consenting blood donors (n=169; age 19-74 years) detected CAF in the limited range of 2.76 ± 0.95 ng/ml. In sarcopenia patients (diagnosed according to clinical and instrumental standards) mean CAF levels were significantly elevated (p=9.8E10-9; n=73; age 65-87 years) compared to aged matched controls. Of all sarcopenia patients, 40% had elevated, non-overlapping CAF levels compared to controls. Evidence is presented for a pathogenic role of the agrin/neurotrypsin system in a substantial subset of sarcopenia patients. These patients are characterized by elevated CAF blood levels compared to aged-matched healthy volunteers suggesting the identification of an agrin-dependent form of sarcopenia. Elevated CAF levels in a large subpopulation of sarcopenic patients suggest the existence of a specific form of sarcopenia for which CAF could become a biomarker and a new target for therapeutic interventions. The feasibility of this approach was demonstrated by the development of a small molecule capable of inhibiting neurotrypsin in vitro and in vivo.

Detection of muscle wasting in patients with chronic heart failure using C-terminal agrin fragment: results from the Studies Investigating Co-morbidities Aggravating Heart Failure (SICA-HF).

Skeletal muscle wasting affects 20% of patients with chronic heart failure and has serious implications for their activities of daily living. Assessment of muscle wasting is technically challenging. C‐terminal agrin‐fragment (CAF), a breakdown product of the synaptically located protein agrin, has shown early promise as biomarker of muscle wasting. We sought to investigate the diagnostic properties of CAF in muscle wasting among patients with heart failure.

C-Terminal Agrin Fragment - A New Fast Biomarker for Kidney Function in Renal Transplant Recipients

Background: The C-terminal agrin fragment (CAF) is a cleavage product of agrin, the major proteoglycan of the glomerular basement membrane. This article studies if CAF could serve as a biomarker for renal function in renal transplant recipients. Material and Methods: We measured serum CAF and creatinine concentrations and calculated estimated glomerular filtration rate (eGFR) (MDRD) in 96 healthy individuals and in 110 end-stage renal disease patients undergoing kidney transplantation before and after transplantation. Correlation between CAF and creatinine concentrations/eGFR was calculated as within-patient (cWP) and between-patient correlations (cBP). Moreover, we evaluated the association of CAF with delayed graft function (DGF). The diagnostic value of CAF for early detection of DGF compared to creatinine was evaluated by receiver operating characteristics (ROC) analysis. Results: CAF concentrations strongly correlated with creatinine (r = 0.86 (cWP), r = 0.74 (cBP)) and eGFR (MDRD) (r = 0.86 (cWP), r = 0.77 (cBP)). Pre-transplant (pre-Tx) CAF concentrations were 19-fold higher than in healthy individuals (1,115.0 (258.4-3,990.0) vs. 56.6 (20.0-109.5) pM). After transplantation, CAF decreased significantly faster than creatinine (postoperative days 1-3 (POD 1-3): 562.8 (101.6-2,113.0) pM; creatinine: pre-Tx 6.9 (3.1-15.7), POD 1-3: 6.4 (1.7-12.7) mg/dl, p < 0.001). Stable concentrations were reached 1-3 months after transplantation for CAF and creatinine (CAF 145.1 (6.7-851.0) pM; creatinine 1.6 (0.7-8.0) mg/dl). CAF concentrations at POD 1-3 were significantly associated with DGF and outperformed creatinine in early detection of DGF (area under the curve (AUC) CAF 80.7% (95% CI 72.3-89.1%) vs. AUC creatinine 71.3% (95% CI 61.8-81.1%), p = 0.061). Conclusion: CAF is a promising new and fast biomarker for kidney function and may serve as a new tool for the early detection of DGF.

Injection of a Soluble Fragment of Neural Agrin (NT-1654) Considerably Improves the Muscle Pathology Caused by the Disassembly of the Neuromuscular Junction

Treatment of neuromuscular diseases is still an unsolved problem. Evidence over the last years strongly indicates the involvement of malformation and dysfunction of neuromuscular junctions in the development of such medical conditions. Stabilization of NMJs thus seems to be a promising approach to attenuate the disease progression of muscle wasting diseases. An important pathway for the formation and maintenance of NMJs is the agrin/Lrp4/MuSK pathway. Here we demonstrate that the agrin biologic NT-1654 is capable of activating the agrin/Lrp4/MuSK system in vivo, leading to an almost full reversal of the sarcopenia-like phenotype in neurotrypsin-overexpressing (SARCO) mice. We also show that injection of NT-1654 accelerates muscle re-innervation after nerve crush. This report demonstrates that a systemically administered agrin fragment has the potential to counteract the symptoms of neuromuscular disorders.

Denervation drives mitochondrial dysfunction in skeletal muscle of octogenarians

Mitochondria are frequently implicated in the ageing of skeletal muscle, although the role of denervation in modulating mitochondrial function in ageing muscle is unknown. We show that increased sensitivity to apoptosis initiation occurs prior to evidence of persistent denervation and is thus a primary mitochondrial defect in ageing muscle worthy of therapeutic targeting. However, at more advanced age, mitochondrial function changes are markedly impacted by persistent sporadic myofibre denervation, suggesting the mitochondrion may be a less viable therapeutic target.

Evaluation of C-terminal Agrin Fragment as a marker of muscle wasting in patients after acute stroke during early rehabilitation.

C‐terminal Agrin Fragment (CAF) has been proposed as a novel biomarker for sarcopenia originating from the degeneration of the neuromuscular junctions. In patients with stroke muscle wasting is a common observation that predicts functional outcome. We aimed to evaluate agrin sub‐fragment CAF22 as a marker of decreased muscle mass and physical performance in the early phase after acute stroke.

C-Terminal Fragment of Agrin (CAF): A Novel Marker for Progression of Kidney Disease in Type 2 Diabetics

Background Diabetes is the leading cause of CKD in the developed world. C-terminal fragment of agrin (CAF) is a novel kidney function and injury biomarker. We investigated whether serum CAF predicts progression of kidney disease in type 2 diabetics. Methods Serum CAF levels were measured in 71 elderly patients with diabetic nephropathy using a newly developed commercial ELISA kit (Neurotune®). Estimated glomerular filtration rate (eGFR) and proteinuria in spot urine were assessed at baseline and after 12 months follow up. The presence of end stage renal disease (ESRD) was evaluated after 24 months follow-up. Correlation and logistic regression analyses were carried out to explore the associations of serum CAF levels with GFR, proteinuria, GFR loss and incident ESRD. Renal handling of CAF was tested in neurotrypsin-deficient mice injected with recombinant CAF. Results We found a strong association of serum CAF levels with eGFR and a direct association with proteinuria both at baseline (r = 0.698, p<0.001 and r = 0. 287, p = 0.02) as well as after 12 months follow-up (r = 0.677, p<0.001 and r = 0.449, p<0.001), respectively. Furthermore, in multivariate analysis, serum CAF levels predicted eGFR decline at 12 months follow-up after adjusting for known risk factors (eGFR, baseline proteinuria) [OR (95%CI) = 4.2 (1.2–14.5), p = 0.024]. In mice, injected CAF was detected in endocytic vesicles of the proximal tubule. Conclusion Serum CAF levels reflect renal function and are highly associated with eGFR and proteinuria at several time points. Serum CAF was able to predict subsequent loss of renal function irrespective of baseline proteinuria in diabetic nephropathy. CAF is likely removed from circulation by glomerular filtration and subsequent endocytosis in the proximal tubule. These findings may open new possibilities for clinical trial design, since serum CAF levels may be used as a selection tool to monitor kidney function in high-risk patients with diabetic nephropathy.