Progranulin functions as a cathepsin D chaperone to stimulate axonal outgrowth in vivo.

Loss of function mutations in progranulin (GRN) cause frontotemporal dementia, but how GRN haploinsufficiency causes neuronal dysfunction remains unclear. We previously showed that GRN is neurotrophic in vitro. Here, we used an in vivo axonal outgrowth system and observed a delayed recovery in GRN-/- mice after facial nerve injury. This deficit was rescued by reintroduction of human GRN and relied on its C-terminus and on neuronal GRN production. Transcriptome analysis of the facial motor nucleus post injury identified cathepsin D (CTSD) as the most upregulated gene. In aged GRN-/- cortices, CTSD was also upregulated, but the relative CTSD activity was reduced and improved upon exogenous GRN addition. Moreover, GRN and its C-terminal granulin domain granulinE (GrnE) both stimulated the proteolytic activity of CTSD in vitro. Pull-down experiments confirmed a direct interaction between GRN and CTSD. This interaction was also observed with GrnE and stabilized the CTSD enzyme at different temperatures. Investigating the importance of this interaction for axonal regeneration in vivo we found that, although individually tolerated, a combined reduction of GRN and CTSD synergistically reduced axonal outgrowth. Our data links the neurotrophic effect of GRN and GrnE with a lysosomal chaperone function on CTSD to maintain its proteolytic capacity.

Microglia lacking a peroxisomal ß-oxidation enzyme chronically alter their inflammatory profile without evoking neuronal and behavioral deficits.

BACKGROUND: Microglia play a central role in most neurological disorders, but the impact of microgliosis on brain environment and clinical functions is not fully understood. Mice lacking multifunctional protein-2 (MFP2), a pivotal enzyme in peroxisomal ß-oxidation, develop a fatal disorder characterized by motor problems similar to the milder form of MFP2 deficiency in humans. The hallmark of disease in mice is the chronic proliferation of microglia in the brain, but molecular pathomechanisms that drive rapid clinical deterioration in human and mice remain unknown. In the present study, we identified the effects of specific deletion of MFP2 from microglia in the brain on immune responses, neuronal functioning, and behavior. METHODS: We created a novel Cx3cr1-Mfp2-/- mouse model and studied the impact of MFP2 deficiency on microglial behavior at different ages using immunohistochemistry and real-time PCR. Pro- and anti-inflammatory responses of Mfp2-/- microglia were assessed in vitro and in vivo after stimulation with IL-1ß/INFγ and IL-4 (in vitro) and LPS and IL-4 (in vivo). Facial nerve axotomy was unilaterally performed in Cx3cr1-Mfp2-/- and control mice, and microglial functioning in response to neuronal injury was subsequently analyzed by histology and real-time PCR. Finally, neuronal function, motor function, behavior, and cognition were assessed using brainstem auditory evoked potentials, grip strength and inverted grid test, open field exploration, and passive avoidance learning, respectively. RESULTS: We found that Mfp2-/- microglia in a genetically intact brain environment adopt an inflammatory activated and proliferative state. In addition, we found that acute inflammatory and neuronal injury provoked normal responses of Mfp2-/- microglia in Cx3cr1-Mfp2-/- mice during the post-injury period. Despite chronic pro-inflammatory microglial reactivity, Cx3cr1-Mfp2-/- mice exhibited normal neuronal transmission, clinical performance, and cognition. CONCLUSION: Our data demonstrate that MFP2 deficiency in microglia causes intrinsic dysregulation of their inflammatory profile, which is not harmful to neuronal function, motor function, and cognition in mice during their first year of life.

SorCS2 binds progranulin to regulate motor neuron development.

Motor neuron (MN) development and nerve regeneration requires orchestrated action of a vast number of molecules. Here, we identify SorCS2 as a progranulin (PGRN) receptor that is required for MN diversification and axon outgrowth in zebrafish and mice. In zebrafish, SorCS2 knockdown also affects neuromuscular junction morphology and fish motility. In mice, SorCS2 and PGRN are co-expressed by newborn MNs from embryonic day 9.5 until adulthood. Using cell-fate tracing and nerve segmentation, we find that SorCS2 deficiency perturbs cell-fate decisions of brachial MNs accompanied by innervation deficits of posterior nerves. Additionally, adult SorCS2 knockout mice display slower motor nerve regeneration. Interestingly, primitive macrophages express high levels of PGRN, and their interaction with SorCS2-positive motor axon is required during axon pathfinding. We further show that SorCS2 binds PGRN to control its secretion, signaling, and conversion into granulins. We propose that PGRN-SorCS2 signaling controls MN development and regeneration in vertebrates.

Progranulin reduces insoluble TDP-43 levels, slows down axonal degeneration and prolongs survival in mutant TDP-43 mice.

BACKGROUND: TAR DNA binding protein 43 (TDP-43) is the main disease protein in most patients with amyotrophic lateral sclerosis (ALS) and about 50% of patients with frontotemporal dementia (FTD). TDP-43 pathology is not restricted to patients with missense mutations in TARDBP, the gene encoding TDP-43, but also occurs in ALS/FTD patients without known genetic cause or in patients with various other ALS/FTD gene mutations. Mutations in progranulin (GRN), which result in a reduction of ~ 50% of progranulin protein (PGRN) levels, cause FTD with TDP-43 pathology. How loss of PGRN leads to TDP-43 pathology and whether or not PGRN expression protects against TDP-43-induced neurodegeneration is not yet clear. METHODS: We studied the effect of PGRN on the neurodegenerative phenotype in TDP-43(A315T) mice. RESULTS: PGRN reduced the levels of insoluble TDP-43 and histology of the spinal cord revealed a protective effect of PGRN on the loss of large axon fibers in the lateral horn, the most severely affected fiber pool in this mouse model. Overexpression of PGRN significantly slowed down disease progression, extending the median survival by approximately 130 days. A transcriptome analysis did not point towards a single pathway affected by PGRN, but rather towards a pleiotropic effect on different pathways. CONCLUSION: Our findings reveal an important role of PGRN in attenuating mutant TDP-43-induced neurodegeneration.

The neurotrophic properties of progranulin depend on the granulin E domain but do not require sortilin binding.

Progranulin (PGRN) is a growth factor involved in wound healing, inflammation, tumor growth, and neurodegeneration. Mutations in the gene encoding PGRN give rise to shortage of PGRN and cause familial frontotemporal lobar degeneration. PGRN exerts neurotrophic functions and binding of PGRN to the membrane receptor sortilin (SORT1) mediates the endocytosis of PGRN. SORT1-mediated uptake plays an important role in the regulation of extracellular PGRN levels. We studied the role of SORT1 in PGRN-mediated neuroprotection in vitro and in vivo. The survival-enhancing effect of PGRN seemed to be dependent on the granulin E (GRN E) domain. Pharmacologic inhibition of the GRN E-SORT1 interaction or deletion of the SORT1 binding site of GRN E did not abolish its neurotrophic function. In addition, the in vivo phenotype of PGRN knockdown in zebrafish embryos was not phenocopied by SORT1 knockdown. These results suggest that GRN E mediates the neurotrophic properties of PGRN and that binding to SORT1 is not required for this effect.