The amyloid precursor protein (APP) and its neurotoxic cleavage product Aβ are key players in the development of Alzheimer's disease (AD) and appear to be essential for neuronal development and cell homeostasis. Proteolytic processing of APP and its physiological function depend on its interaction with heparin and are influenced by the binding of metal ions and sorLA. We created various mutations of metal binding site M1 residing within the extracellular E2 domain of APP. Using isothermal titration calorimetry and circular dichroism spectroscopy, we analyzed the binding of Cu(2+) and Zn(2+) to APP E2 and identified two mutations that are most suited for functional studies to dissect ion specific effects of metal binding. The H313A mutation abrogates only copper-based effects, whereas the H382A mutation weakens any metal binding at M1 of APP E2. Subsequently, we tested the effect of Cu(2+) and Zn(2+) on the binding of heparin and sorLA to APP E2 using a chromatographic technique and surface plasmon resonance. We show that Zn(2+) and to a larger degree also Cu(2+) enhance the binding of heparin to APP E2, consistent with an extracellular regulation of the function of APP by both metal ions. In contrast, neither ion seemed to affect the interaction between APP E2 and sorLA. This supports an intracellular interaction between the latter two partners that would not sense extracellular variations of metal ions upon synaptic activity.

The human epidermal growth factor receptor 2 (HER2) is an oncogene targeted by several kinase inhibitors and therapeutic antibodies. While the endosomal trafficking of many other receptor tyrosine kinases is known to regulate their oncogenic signalling, the prevailing view on HER2 is that this receptor is predominantly retained on the cell surface. Here, we find that sortilin-related receptor 1 (SORLA; SORL1) co-precipitates with HER2 in cancer cells and regulates HER2 subcellular distribution by promoting recycling of the endosomal receptor back to the plasma membrane. SORLA protein levels in cancer cell lines and bladder cancers correlates with HER2 levels. Depletion of SORLA triggers HER2 targeting to late endosomal/lysosomal compartments and impairs HER2-driven signalling and in vivo tumour growth. SORLA silencing also disrupts normal lysosome function and sensitizes anti-HER2 therapy sensitive and resistant cancer cells to lysosome-targeting cationic amphiphilic drugs. These findings reveal potentially important SORLA-dependent endosomal trafficking-linked vulnerabilities in HER2-driven cancers.

Recombinant adeno-associated virus (rAAV) vectors have emerged as the safe vehicles of choice for long-term gene transfer in mammalian nervous system. Recombinant adeno-associated virus-mediated localized gene transfer in adult nervous system following direct inoculation, that is, intracerebral or intrathecal, is well documented. However, recombinant adeno-associated virus delivery in defined neuronal populations in adult animals using less-invasive methods as well as avoiding ectopic gene expression following systemic inoculation remain challenging. Harnessing the capability of some recombinant adeno-associated virus serotypes for retrograde transduction may potentially address such limitations (Note: The term retrograde transduction in this manuscript refers to the uptake of injected recombinant adeno-associated virus particles at nerve terminals, retrograde transport, and subsequent transduction of nerve cell soma). In some studies, recombinant adeno-associated virus serotypes 2/6, 2/8, and 2/9 have been shown to exhibit transduction of connected neuroanatomical tracts in adult animals following lower limb intramuscular recombinant adeno-associated virus delivery in a pattern suggestive of retrograde transduction. However, an extensive side-by-side comparison of these serotypes following intramuscular delivery regarding tissue viral load, and the effect of promoter on transgene expression, has not been performed. Hence, we delivered recombinant adeno-associated virus serotypes 2/6, 2/8, or 2/9 encoding enhanced green fluorescent protein (eGFP), under the control of either cytomegalovirus (CMV) or human synapsin (hSyn) promoter, via a single unilateral hindlimb intramuscular injection in the bicep femoris of adult C57BL/6J mice. Four weeks post injection, we quantified viral load and transgene (enhanced green fluorescent protein) expression in muscle and related nervous tissues. Our data show that the select recombinant adeno-associated virus serotypes transduce sciatic nerve and groups of neurons in the dorsal root ganglia on the injected side, indicating that the intramuscular recombinant adeno-associated virus delivery is useful for achieving gene transfer in local neuroanatomical tracts. We also observed sparse recombinant adeno-associated virus viral delivery or eGFP transduction in lumbar spinal cord and a noticeable lack thereof in brain. Therefore, further improvements in recombinant adeno-associated virus design are warranted to achieve efficient widespread retrograde transduction following intramuscular and possibly other peripheral routes of delivery.

SORL1 is strongly associated with both sporadic and familial forms of Alzheimer's disease (AD), but a lack of information about alternatively spliced transcripts currently limits our understanding of the role of SORL1 in AD. Here, we describe a SORL1 transcript (SORL1-38b) characterized by inclusion of a novel exon (E38b) that encodes a truncated protein. We identified E38b-containing transcripts in several brain regions, with the highest expression in the cerebellum and showed that SORL1-38b is largely located in neuronal dendrites, which is in contrast to the somatic distribution of transcripts encoding the full-length SORLA protein (SORL1-fl). SORL1-38b transcript levels were significantly reduced in AD cerebellum in three independent cohorts of postmortem brains, whereas no changes were observed for SORL1-fl. A trend of lower 38b transcript level in cerebellum was found for individuals carrying the risk variant at rs2282649 (known as SNP24), although not reaching statistical significance. These findings suggest synaptic functions for SORL1-38b in the brain, uncovering novel aspects of SORL1 that can be further explored in AD research.

The established causal genes in Alzheimer's disease (AD), APP, PSEN1, and PSEN2, are functionally characterized using biomarkers, capturing an in vivo profile reflecting the disease's initial preclinical phase. Mutations in SORL1, encoding the endosome recycling receptor SORLA, are found in 2%-3% of individuals with early-onset AD, and SORL1 haploinsufficiency appears to be causal for AD. To test whether SORL1 can function as an AD causal gene, we use CRISPR-Cas9-based gene editing to develop a model of SORL1 haploinsufficiency in Göttingen minipigs, taking advantage of porcine models for biomarker investigations. SORL1 haploinsufficiency in young adult minipigs is found to phenocopy the preclinical in vivo profile of AD observed with APP, PSEN1, and PSEN2, resulting in elevated levels of β-amyloid (Aβ) and tau preceding amyloid plaque formation and neurodegeneration, as observed in humans. Our study provides functional support for the theory that SORL1 haploinsufficiency leads to endosome cytopathology with biofluid hallmarks of autosomal dominant AD.

Whether and how the pathogenic disruptions in endosomal trafficking observed in Alzheimer's disease (AD) are linked to its anatomical vulnerability remain unknown. Here, we began addressing these questions by showing that neurons are enriched with a second retromer core, organized around VPS26b, differentially dedicated to endosomal recycling. Next, by imaging mouse models, we show that the trans-entorhinal cortex, a region most vulnerable to AD, is most susceptible to VPS26b depletion-a finding validated by electrophysiology, immunocytochemistry, and behavior. VPS26b was then found enriched in the trans-entorhinal cortex of human brains, where both VPS26b and the retromer-related receptor SORL1 were found deficient in AD. Finally, by regulating glutamate receptor and SORL1 recycling, we show that VPS26b can mediate regionally selective synaptic dysfunction and SORL1 deficiency. Together with the trans-entorhinal's unique network properties, hypothesized to impose a heavy demand on endosomal recycling, these results suggest a general mechanism that can explain AD's regional vulnerability.

The SORL1 gene has recently emerged as a strong Alzheimer's Disease (AD) risk gene. Over 500 different variants have been identified in the gene and the contribution of individual variants to AD development and progression is still largely unknown. Here, we describe a family consisting of 2 parents and 5 offspring. Both parents were affected with dementia and one had confirmed AD pathology with an age of onset >75 years. All offspring were affected with AD with ages at onset ranging from 53yrs-74yrs. DNA was available from the parent with confirmed AD and 5 offspring. We identified a coding variant, p.(Arg953Cys), in SORL1 in 5 of 6 individuals affected by AD. Notably, variant carriers had severe AD pathology, and the SORL1 variant segregated with TDP-43 pathology (LATE-NC). We further characterized this variant and show that this Arginine substitution occurs at a critical position in the YWTD-domain of the SORL1 translation product, SORL1. Functional studies further show that the p.R953C variant leads to retention of the SORL1 protein in the endoplasmic reticulum which leads to decreased maturation and shedding of the receptor and prevents its normal endosomal trafficking. Together, our analysis suggests that p.R953C is a pathogenic variant of SORL1 and sheds light on mechanisms of how missense SORL1 variants may lead to AD.

Circumstantial evidence points to a pathological role of alpha-synuclein (aSyn; gene symbol SNCA), conferred by aSyn misfolding and aggregation, in Parkinson disease (PD) and related synucleinopathies. Several findings in experimental models implicate perturbations in the tissue homeostatic mechanisms triggered by pathological aSyn accumulation, including impaired redox homeostasis, as significant contributors in the pathogenesis of PD. The nuclear factor erythroid 2-related factor (NRF2/Nrf2) is recognized as 'the master regulator of cellular anti-oxidant response', both under physiological as well as in pathological conditions. Using immunohistochemical analyses, we show a robust nuclear NRF2 accumulation in post-mortem PD midbrain, detected by NRF2 phosphorylation on the serine residue 40 (nuclear active p-NRF2, S40). Curated gene expression analyses of four independent publicly available microarray datasets revealed considerable alterations in NRF2-responsive genes in the disease affected regions in PD, including substantia nigra, dorsal motor nucleus of vagus, locus coeruleus and globus pallidus. To further examine the putative role of pathological aSyn accumulation on nuclear NRF2 response, we employed a transgenic mouse model of synucleionopathy (M83 line, expressing the mutant human A53T aSyn), which manifests widespread aSyn pathology (phosphorylated aSyn; S129) in the nervous system following intramuscular inoculation of exogenous fibrillar aSyn. We observed strong immunodetection of nuclear NRF2 in neuronal populations harboring p-aSyn (S129), and found an aberrant anti-oxidant and inflammatory gene response in the affected neuraxis. Taken together, our data support the notion that pathological aSyn accumulation impairs the redox homeostasis in nervous system, and boosting neuronal anti-oxidant response is potentially a promising approach to mitigate neurodegeneration in PD and related diseases.

The SORL1 gene has recently emerged as a strong Alzheimer's Disease (AD) risk gene. Over 500 different variants have been identified in the gene and the contribution of individual variants to AD development and progression is still largely unknown. Here, we describe a family consisting of 2 parents and 5 offspring. Both parents were affected with dementia and one had confirmed AD pathology with an age of onset > 75 years. All offspring were affected with AD with ages at onset ranging from 53 years to 74 years. DNA was available from the parent with confirmed AD and 5 offspring. We identified a coding variant, p.(Arg953Cys), in SORL1 in 5 of 6 individuals affected by AD. Notably, variant carriers had severe AD pathology, and the SORL1 variant segregated with TDP-43 pathology (LATE-NC). We further characterized this variant and show that this Arginine substitution occurs at a critical position in the YWTD-domain of the SORL1 translation product, SORL1. Functional studies further show that the p.R953C variant leads to retention of the SORL1 protein in the endoplasmic reticulum which leads to decreased maturation and shedding of the receptor and prevents its normal endosomal trafficking. Together, our analysis suggests that p.R953C is a pathogenic variant of SORL1 and sheds light on mechanisms of how missense SORL1 variants may lead to AD.

Recent genome-wide association studies (GWAS) have revealed strong association of hypercholesterolemia and myocardial infarction with SNPs on human chromosome 1p13.3. This locus covers three genes: SORT1, CELSR2, and PSRC1. We demonstrate that sortilin, encoded by SORT1, is an intracellular sorting receptor for apolipoprotein (apo) B100. It interacts with apoB100 in the Golgi and facilitates the formation and hepatic export of apoB100-containing lipoproteins, thereby regulating plasma low-density lipoprotein (LDL) cholesterol. Absence of sortilin in gene-targeted mice reduces secretion of lipoproteins from the liver and ameliorates hypercholesterolemia and atherosclerotic lesion formation in LDL receptor-deficient animals. In contrast, sortilin overexpression stimulates hepatic release of lipoproteins and increases plasma LDL levels. Our data have uncovered a regulatory pathway in hepatic lipoprotein export and suggest a molecular explanation for the cardiovascular risk being associated with 1p13.3.

Huntington's disease (HD) is a dominantly inherited neurological disorder caused by CAG-repeat expansion in exon 1 of Huntingtin (HTT). But in addition to the neurological disease, mutant HTT (mHTT), which is ubiquitously expressed, impairs other organ systems. Indeed, epidemiological and animal model studies suggest higher incidence of and mortality from heart disease in HD. Here, we show that the protein complex mTORC1 is dysregulated in two HD mouse models through a mechanism that requires intrinsic mHTT expression. Moreover, restoring cardiac mTORC1 activity with constitutively active Rheb prevents mortality and relieves the mHTT-induced block to hypertrophic adaptation to cardiac stress. Finally, we show that chronic mTORC1 dysregulation is due in part to mislocalization of endogenous Rheb. These data provide insight into the increased cardiac-related mortality of HD patients, with cardiac mHTT expression inhibiting mTORC1 activity, limiting heart growth, and decreasing the heart's ability to compensate to chronic stress.

Loss of the Sortilin-related receptor 1 (SORL1) gene seems to act as a causal event for Alzheimer's disease (AD). Recent studies have established that loss of SORL1, as well as mutations in autosomal dominant AD genes APP and PSEN1/2, pathogenically converge by swelling early endosomes, AD's cytopathological hallmark. Acting together with the retromer trafficking complex, SORL1 has been shown to regulate the recycling of the amyloid precursor protein (APP) out of the endosome, contributing to endosomal swelling and to APP misprocessing. We hypothesized that SORL1 plays a broader role in neuronal endosomal recycling and used human induced pluripotent stem cell-derived neurons (hiPSC-Ns) to test this hypothesis. We examined endosomal recycling of three transmembrane proteins linked to AD pathophysiology: APP, the BDNF receptor Tropomyosin-related kinase B (TRKB), and the glutamate receptor subunit AMPA1 (GLUA1).

Huntington's Disease (HD) is a dominantly inherited neurodegenerative disease for which the major causes of mortality are neurodegeneration-associated aspiration pneumonia followed by cardiac failure. mTORC1 pathway perturbations are present in HD models and human tissues. Amelioration of mTORC1 deficits by genetic modulation improves disease phenotypes in HD models, is not a viable therapeutic strategy. Here, we assessed a novel small molecule mTORC1 pathway activator, NV-5297, for its improvement of the disease phenotypes in the N171-82Q HD mouse model. Oral dosing of NV-5297 over 6 weeks activated mTORC1, increased striatal volume, improved motor learning and heart contractility. Further, the heart contractility, heart fibrosis, and survival were improved in response to the cardiac stressor isoprenaline when compared to vehicle-treated mice. Cummulatively, these data support mTORC1 activation as a therapeutic target in HD and consolidates NV-5297 as a promising drug candidate for treating central and peripheral HD phenotypes and, more generally, mTORC1-deficit related diseases.

Current evidence indicates that resistance to the tyrosine kinase-type cell surface receptor (HER2)-targeted therapies is frequently associated with HER3 and active signaling via HER2-HER3 dimers, particularly in the context of breast cancer. Thus, understanding the response to HER2-HER3 signaling and the regulation of the dimer is essential to decipher therapy relapse mechanisms. Here, we investigate a bidirectional relationship between HER2-HER3 signaling and a type-1 transmembrane sorting receptor, sortilin-related receptor (SorLA; SORL1). We demonstrate that heregulin-mediated signaling supports SorLA transcription downstream of the mitogen-activated protein kinase pathway. In addition, we demonstrate that SorLA interacts directly with HER3, forming a trimeric complex with HER2 and HER3 to attenuate lysosomal degradation of the dimer in a Ras-related protein Rab4-dependent manner. In line with a role for SorLA in supporting the stability of the HER2 and HER3 receptors, loss of SorLA compromised heregulin-induced cell proliferation and sensitized metastatic anti-HER2 therapy-resistant breast cancer cells to neratinib in cancer spheroids in vitro and in vivo in a zebrafish brain xenograft model.

Schwann cell reprogramming and differentiation are crucial prerequisites for neuronal regeneration and re-myelination to occur following injury to peripheral nerves. The neurotrophin receptor p75NTR has been identified as a positive modulator for Schwann cell myelination during development and implicated in promoting nerve regeneration after injury. However, most studies base this conclusion on results obtained from complete p75NTR knockout mouse models and cannot dissect the specific role of p75NTR expressed by Schwann cells. In this present study, a conditional knockout model selectively deleting p75NTR expression in Schwann cells was generated, where p75NTR expression is replaced with that of an mCherry reporter. Silencing of Schwann cell p75NTR expression was confirmed in the sciatic nerve in vivo and in vitro, without altering axonal expression of p75NTR. No difference in sciatic nerve myelination during development or following sciatic nerve crush injury was observed, as determined by quantification of both myelinated and unmyelinated nerve fiber densities, myelinated axonal diameter and myelin thickness. However, the absence of Schwann cell p75NTR reduced motor nerve conduction velocity after crush injury. Our data indicate that the absence of Schwann cell p75NTR expression in vivo is not critical for axonal regrowth or remyelination following sciatic nerve crush injury, but does play a key role in functional recovery. Overall, this represents the first step in redefining the role of p75NTR in the peripheral nervous system, suggesting that the Schwann cell-axon unit functions as a syncytium, with the previous published involvement of p75NTR in remyelination most likely depending on axonal/neuronal p75NTR and/or mutual glial-axonal interactions.

Schwann cells (SCs) are the main glial cells of the peripheral nervous system (PNS) and are known to be involved in various pathophysiological processes, such as diabetic neuropathy and nerve regeneration, through neurotrophin signaling. Such glial trophic support to axons, as well as neuronal survival/death signaling, has previously been linked to the p75 neurotrophin receptor (p75NTR) and its co-receptor Sortilin. Recently, SC-derived extracellular vesicles (EVs) were shown to be important for axon growth and nerve regeneration, but cargo of these glial cell-derived EVs has not yet been well-characterized. In this study, we aimed to characterize signatures of small RNAs in EVs derived from wild-type (WT) SCs and define differentially expressed small RNAs in EVs derived from SCs with genetic deletions of p75NTR (Ngfr-/-) or Sortilin (Sort1-/-). Using RNA sequencing, we identified a total of 366 miRNAs in EVs derived from WT SCs of which the most highly expressed are linked to the regulation of axonogenesis, axon guidance and axon extension, suggesting an involvement of SC EVs in axonal homeostasis. Signaling of SC EVs to non-neuronal cells was also suggested by the presence of several miRNAs important for regulation of the endothelial cell apoptotic process. Ablated p75NTR or sortilin expression in SCs translated into a set of differentially regulated tRNAs and miRNAs, with impact in autophagy and several cellular signaling pathways such as the phosphatidylinositol signaling system. With this work, we identified the global expression profile of small RNAs present in SC-derived EVs and provided evidence for a regulatory function of these vesicles on the homeostasis of other cell types of the PNS. Differentially identified miRNAs can pave the way to a better understanding of p75NTR and sortilin roles regarding PNS homeostasis and disease.

The ε4 allele of apolipoprotein E (APOE) is the strongest genetic risk factor for sporadic Alzheimer's Disease (AD). Knockdown of this allele may provide a therapeutic strategy for AD, but the effect of APOE loss-of-function (LoF) on AD pathogenesis is unknown. We searched for APOE LoF variants in a large cohort of older controls and patients with AD and identified six heterozygote carriers of APOE LoF variants. Five carriers were controls (ages 71-90) and one was an AD case with an unremarkable age-at-onset between 75-79. Two APOE ε3/ε4 controls (Subjects 1 and 2) carried a stop-gain affecting the ε4 allele. Subject 1 was cognitively normal at 90+ and had no neuritic plaques at autopsy. Subject 2 was cognitively healthy within the age range 75-79 and underwent lumbar puncture at between ages 75-79 with normal levels of amyloid. The results provide the strongest human genetics evidence yet available suggesting that ε4 drives AD risk through a gain of abnormal function and support knockdown of APOE ε4 or its protein product as a viable therapeutic option.

SORL1, the gene encoding the large multidomain SORLA protein, has emerged as only the fourth gene that when mutated can by itself cause Alzheimer's disease (AD), and as a gene reliably linked to both the early- and late-onset forms of the disease. SORLA is known to interact with the endosomal trafficking regulatory complex called retromer in regulating the recycling of endosomal cargo, including the amyloid precursor protein (APP) and the glutamate receptor GluA1. Nevertheless, SORLA's precise structural-functional relationship in endosomal recycling tubules remains unknown. Here, we address these outstanding questions by relying on crystallographic and artificial-intelligence evidence to generate a structural model for how SORLA folds and fits into retromer-positive endosomal tubules, where it is found to dimerize via both SORLA's fibronectin-type-III (3Fn)- and VPS10p-domains. Moreover, we identify a SORLA fragment comprising the 3Fn-, transmembrane, and cytoplasmic domains that has the capacity to form a dimer, and to enhance retromer-dependent recycling of APP by decreasing its amyloidogenic processing. Collectively, these observations generate a model for how SORLA dimer (and possibly polymer) formation can function in stabilizing and enhancing retromer function at endosome tubules. These findings can inform investigation of the many AD-associated SORL1 variants for evidence of pathogenicity and can guide discovery of novel drugs for the disease.

Peripheral nerve regeneration relies on the ability of Schwann cells to support the regrowth of damaged axons. Schwann cells re-differentiate when reestablishing contact with the sprouting axons, with large fibers becoming remyelinated and small nociceptive fibers ensheathed and collected into Remak bundles. We have previously described how the receptor sortilin facilitates neurotrophin signaling in peripheral neurons via regulated trafficking of Trk receptors. This study aims to characterize the effects of sortilin deletion on nerve regeneration following sciatic crush injury. We found that Sort1 - / - mice displayed functional motor recovery like that of WT mice, with no detectable differences in relation to nerve conduction velocities and morphological aspects of myelinated fibers. In contrast, we found abnormal ensheathment of regenerated C-fibers in injured Sort1 - / - mice, demonstrating a role of sortilin for Remak bundle formation following injury. Further studies on Schwann cell signaling pathways showed a significant reduction of MAPK/ERK, RSK, and CREB phosphorylation in Sort1 - / - Schwann cells after stimulation with neurotrophin-3 (NT-3), while Schwann cell migration and myelination remained unaffected. In conclusion, our results demonstrate that loss of sortilin blunts NT-3 signaling in Schwann cells which might contribute to the impaired Remak bundle regeneration after sciatic nerve injury.

Neuropathological observations in neurodegenerative synucleinopathies, including Parkinson disease, implicate a pathological role of α-synuclein accumulation in extranigral sites during the prodromal phase of the disease. In a transgenic mouse model of peripheral-to-central neuroinvasion and propagation of α-synuclein pathology (via hindlimb intramuscular inoculation with exogenous fibrillar α-synuclein: the M83 line, expressing the mutant human Ala53Thr α-synuclein), we studied the development and early-stage progression of α-synuclein pathology in the CNS of non-symptomatic (i.e. freely mobile) mice. By immunohistochemical analyses of phosphroylated α-synuclein on serine residue 129 (p-S129), our data indicate that the incipient stage of pathological α-synuclein propagation could be categorized in distinct phases: (i) initiation phase, whereby α-synuclein fibrillar inoculum induced pathological lesions in pools of premotor and motor neurons of the lumbar spinal cord, as early as 14 days post-inoculation; (ii) early central phase, whereby incipient α-synuclein pathology was predominantly detected in the reticular nuclei of the brainstem; and (iii) late central phase, characterized by additional sites of lesions in the brain including vestibular nuclei, deep cerebellar nuclei and primary motor cortex, with coincidental emergence of a sensorimotor deficit (mild degree of hindlimb clasping). Intriguingly, we also detected progressive α-synuclein pathology in premotor and motor neurons in the thoracic spinal cord, which does not directly innervate the hindlimb, as well as in the oligodendroglia within the white matter tracts of the CNS during this prodromal phase. Collectively, our data provide crucial insights into the spatiotemporal propagation of α-synuclein pathology in the nervous system of this rodent model of α-synucleinopathy following origin in periphery, and present a neuropathological context for the progression from pre-symptomatic stage to an early deficit in sensorimotor coordination. These findings also hint towards a therapeutic window for targeting the early stages of α-synuclein pathology progression in this model, and potentially facilitate the discovery of mechanisms relevant to α-synuclein proteinopathies. In a rodent model of synucleinopathy, Ferreira et al., delineate the spatiotemporal progression of incipient α-synuclein pathology (of peripheral origin) in the CNS. The authors show early affection of brainstem reticular nuclei in non-paralyzed mice, and pathological white matter lesions in relation to the neuronal pathology.