Targeted protein degradation (TPD) using PROteolysis TArgeting Chimeras (PROTACs) is a powerful therapeutic strategy for degrading difficult-to-drug cytosolic proteins in the proteasome. Here, we present a strategy for extracellular TPD by reshaping the interaction between the lysosome sorting receptor sortilin and its ligand progranulin for engineering SORtilin-based lysosome TArgeting Chimeras (SORTACs). SORTACs induce ternary complex formation followed by endocytosis and target degradation. SORTAC activity can be genetically encoded, demonstrated by converting an IgG-binding nanobody to an IgG-degrading nanobody or by chemical conjugation, enabling conversion of therapeutic antibodies from binding their target to driving its degradation. Importantly, we generated PROTAC-like small molecule SORTACs with nanomolar range potency against two inflammatory proteins, the cytokine TNFa and the membrane protein vanin-1. Our results demonstrate that SORTACs constitute a modular platform for rapid generation of degraders of extracellular targets and with the potential to have wide impact in drug discovery.

Attention-deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder affecting 5% of children and 2.5% of adults worldwide. ADHD is considered a polygenic disorder caused by a combination of both common and rare risk variants, each with low individual effect size. The Vps10p domain receptor SorCS2 is involved in neuronal development and synaptic plasticity by modulating brain-derived neurotrophic factor (BDNF) signaling. We here describe the identification and characterization of a heterozygous damaging variant in the SORCS2 gene found in two members of a family with persistent ADHD. The SORCS2 variant results in an arginine to tryptophan substitution in the 10CC region of the extracellular Vps10p domain, leading to aberrant posttranslational receptor processing, subcellular localization and ligand binding. Furthermore, the variant abrogates BDNF signaling in a dominant negative manner. Biochemical analysis of additional rare missense variants from ADHD cohorts suggested that SorCS2 structural stability and function is susceptible to such variation in the Vps10p domain. Our findings provide insights into how low frequency damaging variants in SORCS2 may contribute to the risk of ADHD.

Targeted protein degradation (TPD) using PROteolysis TArgeting Chimeras (PROTACs) is a rapidly emerging therapeutic strategy for difficult-to-drug cytosolic proteins. PROTACs are heterobifunctional small molecules that bridge the target with an E3 ubiquitin ligase, destining it for degradation by the proteasome. They have the potential to be orally available and to act catalytically, switching the pharmacology from occupancy-driven to event-driven (1-3). Here we present a strategy for targeted degradation of extracellular proteins by reshaping the interaction between the broadly expressed lysosome sorting receptor sortilin and its ligand progranulin for engineering SORtilin-based lysosome TArgeting Chimeras (SORTACs). SORTACs induce ternary complex formation with the target and sortilin, followed by endocytosis and lysosomal degradation. SORTAC activity can be genetically encoded as demonstrated by converting an IgG binding nanobody to an IgG degrading nanobody or by chemical conjugation, enabling single step conversion of therapeutic antibodies from binding their target to driving its degradation. Importantly, using structure-based design, we generated small molecule SORTACs against the inflammatory cytokine TNFa with nanomolar range potency and with physicochemical properties like PROTACs. Our results demonstrate that SORTACs constitute a versatile and highly modular platform for rapid generation of degraders of in theory any extracellular target and with the potential to have wide impact in drug discovery. ### Competing Interest Statement CG, JV, MK, DK, JL, CL, AS, AG, DO, SMMM, DG, AQ, PG, GW, KTJ, SFN, PM, SG are all current or former employees at Draupnir Bio. The remaining authors have no financial interest related to the present study.

PCSK9 induces lysosomal degradation of the low-density lipoprotein (LDL) receptor (LDLR) in the liver, hereby preventing removal of LDL cholesterol from the circulation. Accordingly, PCSK9 inhibitory antibodies and siRNA potently reduce LDL cholesterol to unprecedented low levels and are approved for treatment of hypercholesterolemia. In addition, PCSK9 inactivation alters the levels of several other circulating lipid classes and species. Brain function is critically influenced by cholesterol and lipid composition. However, it remains unclear how the brain is affected long-term by the reduction in circulating lipids as achieved with potent lipid lowering therapeutics such as PCSK9 inhibitors. Furthermore, it is unknown if locally expressed PCSK9 affects neuronal circuits through regulation of receptor levels. We have studied the effect of lifelong low peripheral cholesterol levels on brain lipid composition and behavior in adult PCSK9 KO mice. In addition, we studied the effect of PCSK9 on neurons in culture and in vivo in the developing cerebral cortex. We found that PCSK9 reduced LDLR and neurite complexity in cultured neurons, but neither PCSK9 KO nor overexpression affected cortical development in vivo . Interestingly, PCSK9 deficiency resulted in changes of several lipid classes in the adult cortex and cerebellum. Despite the observed changes, PCSK9 KO mice had unchanged behavior compared to WT controls. In conclusion, our findings demonstrate that altered PCSK9 levels do not compromise brain development or function in mice, and are in line with clinical trials showing that PCSK9 inhibitors have no adverse effects on cognitive function.

Proteinuria is a prominent feature of chronic kidney disease. Interventions that reduce proteinuria slow the progression of chronic kidney disease and the associated risk of cardiovascular disease. Here, we propose a mechanistic coupling between proteinuria and proprotein convertase subtilisin/kexin type 9 (PCSK9), a regulator of cholesterol and a therapeutic target in cardiovascular disease. PCSK9 undergoes glomerular filtration and is captured by megalin, the receptor responsible for driving protein reabsorption in the proximal tubule. Accordingly, megalin-deficient mice and patients carrying megalin pathogenic variants (Donnai Barrow syndrome) were characterized by elevated urinary PCSK9 excretion. Interestingly, PCSK9 knockout mice displayed increased kidney megalin while PCSK9 overexpression resulted in its reduction. Furthermore, PCSK9 promoted trafficking of megalin to lysosomes in cultured proximal tubule cells, suggesting that PCSK9 is a negative regulator of megalin. This effect can be accelerated under disease conditions since either genetic destruction of the glomerular filtration barrier in podocin knockout mice or minimal change disease (a common cause of nephrotic syndrome) in patients resulted in enhanced tubular PCSK9 uptake and urinary PCSK9 excretion. Pharmacological PCSK9 inhibition increased kidney megalin while reducing urinary albumin excretion in nephrotic mice. Thus, glomerular damage increases filtration of PCSK9 and concomitantly megalin degradation, resulting in escalated proteinuria.

SORCS2 is one of five proteins that constitute the Vps10p-domain receptor family. Members of this family play important roles in cellular processes linked to neuronal survival, differentiation and function. Genetic and functional studies implicate SORCS2 in cognitive function, as well as in neurodegenerative and psychiatric disorders. DNA damage and DNA repair deficits are linked to ageing and neurodegeneration, and transient neuronal DNA double-strand breaks (DSBs) also occur as a result of neuronal activity. Here, we report a novel role for SORCS2 in DSB formation. We show that SorCS2 loss is associated with elevated DSB levels in the mouse dentate gyrus and that knocking out SORCS2 in a human neuronal cell line increased Topoisomerase IIβ-dependent DSB formation and reduced neuronal viability. Neuronal stimulation had no impact on levels of DNA breaks in vitro, suggesting that the observed differences may not be the result of aberrant neuronal activity in these cells. Our findings are consistent with studies linking the VPS10 receptors and DNA damage to neurodegenerative conditions.

Attention deficit hyperactivity disorder (ADHD) is the most frequently diagnosed neurodevelopmental disorder worldwide. Affected individuals present with hyperactivity, inattention, and cognitive deficits and display a characteristic paradoxical response to drugs affecting the dopaminergic system. However, the underlying pathophysiology of ADHD and how this relates to dopaminergic transmission remains to be fully understood. Sorcs2 −/− mice uniquely recapitulate symptoms reminiscent of ADHD in humans. Here, we show that lack of SorCS2 in mice results in lower sucrose intake, indicating general reward deficits. Using in-vivo recordings, we further find that dopaminergic transmission in the ventral tegmental area (VTA) is shifted towards a more regular firing pattern with marked reductions in the relative occurrence of irregular firing in Sorcs2 −/− mice. This was paralleled by abnormal acute behavioral responses to dopamine receptor agonists, suggesting fundamental differences in dopaminergic circuits and indicating a perturbation in the balance between the activities of the postsynaptic dopamine receptor DRD1 and the presynaptic inhibitory autoreceptor DRD2. Interestingly, the hyperactivity and drug response of Sorcs2 −/− mice were markedly affected by novelty. Taken together, our findings show how loss of a candidate ADHD-risk gene has marked effects on dopaminergic circuit function and the behavioral response to the environment.

Alcohol use disorder (AUD) is characterized by repetitive and uncontrolled intake of alcohol with severe consequences for affected individuals, their families and society as a whole. Numerous studies have implicated brain-derived neurotrophic factor (BDNF) activity in the neurobiology underlying AUD. The BDNF signaling mechanism is complex and depends on two receptor systems, TrkB and p75NTR, which appear to have opposite effects on alcohol seeking behavior in animal models. We recently discovered that the sortilin-related receptor SorCS2 forms complexes with both TrkB and p75NTR and is important for BDNF activity in the developing and adult CNS. Moreover, the SORCS2 gene was recently identified as the top association signal for severity of alcohol withdrawal symptoms. Hence, we speculated that SorCS2 deficient mice would have an altered response to alcohol. The role of SorCS2 in the acute and adapted response to alcohol was therefore investigated by comparing SorCS2 knockout (Sorcs2-/- ) mice to wild type (WT) mice in three paradigms modeling alcohol sensitivity and consumption; alcohol-induced conditioned place preference, two-bottle choice test as well as the behavioral response to alcohol withdrawal. We found that, when compared to the WT mice, (I) Sorcs2-/- mice displayed complete lack of alcohol-induced place preference, (II) when given free choice between water and alcohol, Sorcs2-/- mice consumed less alcohol, and (III) Sorcs2-/- mice showed no handling-induced convulsion in response to alcohol withdrawal following extended alcohol exposure. Taken together, these results show that lack of the alcohol withdrawal risk gene Sorcs2 results in abnormal behavioral response to alcohol in mice. Consequently, SorCS2 may play an important role in the molecular pathways underlying AUD and complications associated with alcohol withdrawal.

Coronary artery disease is the main cause of death worldwide and accelerated by increased plasma levels of cholesterol-rich low-density lipoprotein particles (LDL). Circulating PCSK9 contributes to coronary artery disease by inducing lysosomal degradation of the LDL receptor (LDLR) in the liver and thereby reducing LDL clearance. Here, we show that liver heparan sulfate proteoglycans are PCSK9 receptors and essential for PCSK9-induced LDLR degradation. The heparan sulfate-binding site is located in the PCSK9 prodomain and formed by surface-exposed basic residues interacting with trisulfated heparan sulfate disaccharide repeats. Accordingly, heparan sulfate mimetics and monoclonal antibodies directed against the heparan sulfate-binding site are potent PCSK9 inhibitors. We propose that heparan sulfate proteoglycans lining the hepatocyte surface capture PCSK9 and facilitates subsequent PCSK9:LDLR complex formation. Our findings provide new insights into LDL biology and show that targeting PCSK9 using heparan sulfate mimetics is a potential therapeutic strategy in coronary artery disease.

BACKGROUND:Efforts to promote the cessation of harmful alcohol use are hindered by the affective and physiological components of alcohol withdrawal (AW), which can include life-threatening seizures. Although previous studies of AW and relapse have highlighted the detrimental role of stress, little is known about genetic risk factors. METHODS:We conducted a genome-wide association study of AW symptom count in uniformly assessed subjects with histories of serious AW, followed by additional genotyping in independent AW subjects. RESULTS:The top association signal for AW severity was in sortilin family neurotrophin receptor gene SORCS2 on chromosome 4 (European American meta-analysis n = 1,478, p = 4.3 × 10-9 ). There were no genome-wide significant findings in African Americans (n = 1,231). Bioinformatic analyses were conducted using publicly available high-throughput transcriptomic and epigenomic data sets, showing that in humans SORCS2 is most highly expressed in the nervous system. The identified SORCS2 risk haplotype is predicted to disrupt a stress hormone-modulated regulatory element that has tissue-specific activity in human hippocampus. We used human neural lineage cells to demonstrate in vitro a causal relationship between stress hormone levels and SORCS2 expression, and show that SORCS2 levels in culture are increased upon ethanol exposure and withdrawal. CONCLUSIONS:Taken together, these findings indicate that the pathophysiology of withdrawal may involve the effects of stress hormones on neurotrophic factor signaling. Further investigation of these pathways could produce new approaches to managing the aversive consequences of abrupt alcohol cessation.

This article describes an immunocytochemistry (ICC) method for staining against phosphorylated forms of the kinases Akt (pAkt) and MAPK (pMAPK). Phosphorylation is induced upon their activation by a number stimuli including insulin and brain-derived neurotrophic factor (BDNF), and is prerequisite for a number of cellular processes including cell proliferation and survival [1], [2], [3], [4], [5], [6]. ICC using antibodies raised against specific phosphorylation sites allows cell-type specific and subcellular monitoring of kinase activation. Here, we test how four different antibodies against pAkt and pMAPK, respectively perform in different cell types following insulin or BDNF stimulation using different protocol conditions. We find that phospho-specific-antibodies generally perform better when using Triton X-100 as a permeabilization agent compared to Saponin. In addition, two antibodies against pAkt and two against pMAPK gave a clear increase in signal in cells stimulated with insulin or BDNF compared to the signal obtained in unstimulated cells. These antibodies also performed well when tested with western blotting. Our results illustrate that both the choice of antibody as well as protocol details are critical parameters for successful detection of phosphorylated forms of kinases by ICC. This article includes: •A protocol for subcellular detection of phosphorylated Akt and MAPK.•Validation of 8 antibodies by immunocytochemistry.•Confirmation by western blotting.

Balancing trophic and apoptotic cues is critical for development and regeneration of neuronal circuits. Here we identify SorCS2 as a proneurotrophin (proNT) receptor, mediating both trophic and apoptotic signals in conjunction with p75(NTR). CNS neurons, but not glia, express SorCS2 as a single-chain protein that is essential for proBDNF-induced growth cone collapse in developing dopaminergic processes. SorCS2- or p75(NTR)-deficient in mice caused reduced dopamine levels and metabolism and dopaminergic hyperinnervation of the frontal cortex. Accordingly, both knockout models displayed a paradoxical behavioral response to amphetamine reminiscent of ADHD. Contrary, in PNS glia, but not in neurons, proteolytic processing produced a two-chain SorCS2 isoform that mediated proNT-dependent Schwann cell apoptosis. Sciatic nerve injury triggered generation of two-chain SorCS2 in p75(NTR)-positive dying Schwann cells, with apoptosis being profoundly attenuated in Sorcs2(-/-) mice. In conclusion, we have demonstrated that two-chain processing of SorCS2 enables neurons and glia to respond differently to proneurotrophins.

Glial cell-line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor that has reached clinical trials for Parkinson’s disease. GDNF binds to its coreceptor GFRα1 and signals through the transmembrane receptor tyrosine kinase RET, or RET independently through NCAM or syndecan-3. Whereas the GDNF signaling cascades are well described, cellular turnover and trafficking of GDNF and its receptors remain poorly characterized. Here, we find that SorLA acts as sorting receptor for the GDNF/GFRα1 complex, directing it from the cell surface to endosomes. Through this mechanism, GDNF is targeted to lysosomes and degraded while GFRα1 recycles, creating an efficient GDNF clearance pathway. The SorLA/GFRα1 complex further targets RET for endocytosis but not for degradation, affecting GDNF-induced neurotrophic activities. SorLA-deficient mice display elevated GDNF levels, altered dopaminergic function, marked hyperactivity, and reduced anxiety, all of which are phenotypes related to abnormal GDNF activity. Taken together, these findings establish SorLA as a critical regulator of GDNF activity in the CNS.

This subcellular fractionation protocol is used for separation of cellular organelles based on their density. We have designed and optimized the protocol for separation of subcellular compartments of brain homogenates with focus on the localization and trafficking of transmembrane proteins, but we have also successfully used this protocol for fractionation of other types of tissue. The protocol has two major steps 1) preparation of homogenate from dissected tissue and 2) separation of organelles by centrifugation of homogenates using a continuous sucrose gradient.

BDNF-induced signaling is essential for the development of the central nervous system and critical for plasticity in adults. Mature BDNF signals through TrkB, while its precursor proBDNF employs p75(NTR), resulting in activation of signaling cascades with opposite effects on neuronal survival, growth cone decisions, and synaptic plasticity. Accordingly, variations in the genes encoding BDNF and its receptors sometimes have opposing influences in psychiatric disorders, and despite the vast literature, consensus is lacking about the behavioral consequences of disrupting the activity of the BDNF system in mice. To dissect the behavioral traits affected by dysfunctional BDNF/TrkB vs. proBDNF/p75(NTR) activity, we studied Bdnf(+/-), Ntrk2(+/-), and Ngfr(-/-) mice in parallel with respect to exploratory behavior, anxiety, startle, and fear acquisition. Our data reveal that the effect of proBDNF/BDNF and its receptors on behavior is more complex than expected. Strikingly, receptor-deficient mice displayed increased risk-taking behavior in the open field and elevated plus maze, whereas lack of proBDNF/BDNF had the opposite effect on mouse behavior. On the other hand, although TrkB signaling is instrumental for acquisition of fear memory in an inhibitory avoidance experiment, lack of p75(NTR) or proBDNF/BDNF conferred increased memory in this task. Importantly, none of the genotypes displayed any deficits in startle reflex, indicating unimpaired response to shock. The combined data illustrate an apparent paradox in the role of the BDNF system in controlling complex behavior and suggest that the individual components may also engage independently in separate signaling pathways.

The development and progression of Alzheimer's disease is linked to excessive production of toxic amyloid-β peptide, initiated by β-secretase cleavage of the amyloid precursor protein (APP). In contrast, soluble APPα (sAPPα) generated by the α-secretase is known to stimulate dendritic branching and enhance synaptic function. Regulation of APP processing, and the shift from neurotrophic to neurotoxic APP metabolism remains poorly understood, but the cellular localization of APP and its interaction with various receptors is considered important. We here identify sortilin as a novel APP interaction partner. Like the related APP receptor SorLA, sortilin is highly expressed in the CNS, but whereas SorLA mainly colocalizes with APP in the soma, sortilin interacts with APP in neurites. The presence of sortilin promotes α-secretase cleavage of APP, unlike SorLA, which inhibits the generation of all soluble products. Also, sortilin and SorLA both bind and mediate internalization of sAPP but to different cellular compartments. The interaction involves the 6A domain of APP, present in both neuronal and non-neuronal APP isoforms. This is important as sAPP receptors described so far only bind the non-neuronal isoforms, leaving SorLA and sortilin as the only receptors for sAPP generated by neurons. Together, our findings establish sortilin, as a novel APP interaction partner that influences both production and cellular uptake of sAPP.