A Novel Monoallelic ALG5 Variant Causing Late-Onset ADPKD and Tubulointerstitial Fibrosis.

Introduction: Monoallelic variants in the ALG5 gene encoding asparagine-linked glycosylation protein 5 homolog (ALG5) have been recently shown to disrupt polycystin-1 (PC1) maturation and trafficking via underglycosylation, causing an autosomal dominant polycystic kidney disease-like (ADPKD-like) phenotype and interstitial fibrosis. In this report, we present clinical, genetic, histopathologic, and protein structure and functional correlates of a new ALG5 variant, p.R79W, that we identified in 2 distant genetically related Irish families displaying an atypical late-onset ADPKD phenotype combined with tubulointerstitial damage. Methods: Whole exome and targeted sequencing were used for segregation analysis of available relatives. This was followed by immunohistochemistry examinations of kidney biopsies, and targeted (UMOD, MUC1) and untargeted plasma proteome and N-glycomic studies. Results: We identified a monoallelic ALG5 variant [GRCh37 (NM_013338.5): g.37569565G>A, c.235C>T; p.R79W] that cosegregates in 23 individuals, of whom 18 were clinically affected. We detected abnormal localization of ALG5 in the Golgi apparatus of renal tubular cells in patients' kidney specimens. Further, we detected the pathological accumulation of uromodulin, an N-glycosylated glycosylphosphatidylinositol (GPI)-anchored protein, in the endoplasmic reticulum (ER), but not mucin-1, an O- and N-glycosylated protein. Biochemical investigation revealed decreased plasma and urinary uromodulin levels in clinically affected individuals. Proteomic and glycoproteomic profiling revealed the dysregulation of chronic kidney disease (CKD)-associated proteins. Conclusion: ALG5 dysfunction adversely affects maturation and trafficking of N-glycosylated and GPI anchored protein uromodulin, leading to structural and functional changes in the kidney. Our findings confirm ALG5 as a cause of late-onset ADPKD and provide additional insight into the molecular mechanisms of ADPKD-ALG5.

An unusual case of nephrotic syndrome.

BACKGROUND: Alport syndrome is a genetically heterogenous disorder resulting from variants in genes coding for alpha-3/4/5 chains of Collagen IV, which results in defective basement membranes in the kidney, cochlea and eye. The syndrome has different inheritance patterns and historically, was thought of as a disease affecting solely males. CASE: A 15-year-old female presented with pedal oedema, hypertension and proteinuria. She underwent a kidney biopsy which showed findings in keeping with focal segmental glomerulosclerosis. Her condition was refractory to steroids. Steroid-resistant nephrotic syndrome genetics were sent, revealing a rare pathogenic variant in the COL4A5 gene. CONCLUSION: Heterozygous females with X-linked Alport syndrome can develop chronic kidney disease and hearing loss. Clinicians should be mindful when reviewing kidney histology to include Alport syndrome as a differential for female patients. COL4A3-5 genes should be included in all steroid-resistant nephrotic syndrome genetic panels.

The effects of stocking density on behavior and biological functioning of penned sheep under continuous heat load conditions.

Stocking density may impact sheep welfare during live export voyages that occur under hot and humid conditions. The aim of this study was to assess the welfare implications for sheep housed at three allometric stocking densities (k = 0.030, 0.033, 0.042), while exposed to hot and humid climatic conditions. For 21 d, Merino wethers (n = 216) were housed in 12 pens of 18 wethers, in two climate-controlled rooms where wet-bulb temperature (TWB) mimicked the conditions of a live export voyage with high heat and humidity, and limited diurnal variation. Scan sampling of standing and lying behaviors was conducted on days 2, 5, 8, 11, 15, 18, and 20, at hourly intervals. Agonistic interactions were scored continuously on the same days between 1750 and 1800 h. Liveweights were recorded at the start and end of the study. For a subset of focal wethers (3 per pen), whole blood variables were assessed at the start and end of the experiment, along with fecal glucocorticoid metabolites (FGCM), which were also assessed on days 7 and 14. Rumen temperatures (TRUM) of focal wethers were recorded at 10-min intervals, and their respiration rates (RR) were measured every 2 h on days 1, 3, and from days 7 to 21. Focal wethers were slaughtered for necropsy after the study, and both adrenal glands were excised and weighed. The expression of some lying positions was impaired at high stocking densities, and lying with outstretched legs increased at high TWB. For respiration rates, there was an interaction between stocking density and TWB, such that RR was reduced by the provision of additional space at high TWB. TRUM was relatively unaffected by stocking density but increased at higher TWB, and any effects of stocking density on FGCM concentrations, liveweights (LW), adrenal gland weights or blood variables were minimal. Necropsy examination showed no indication that the wethers had experienced ongoing respiratory distress. These results suggest that the wethers were able to cope with these increases in stocking density under the conditions imposed. However, based on this evidence, the provision of additional space under hot conditions may be beneficial to facilitating the expression of some lying positions. Whilst the experiment was designed to emulate certain conditions relevant during live export voyages, other factors that may induce stress during this mode of transport were not present, and so the conclusions must be interpreted in the context of the experimental conditions.

There is a high demand for Australian sheep to be exported to the Middle East, and for live export voyages which depart during an Australian winter, heat, and humidity increase rapidly as ships cross the equator and approach destination countries. Concern about sheep becoming heat stressed during these voyages has increased, and industry attention has focused on the potential role of stocking density in determining heat stress risk in this context. High stocking densities limit the body surface area available for heat loss and can increase heat exchange between individual sheep. This study aimed to assess the welfare implications of three stocking densities, for sheep exposed to climatic conditions similar to those experienced during a live export voyage to the Middle East. Higher stocking densities restricted the ability of sheep to lie in some positions, but stocking density had limited effects on heat stress indicators or the physiology of the sheep. These results suggested that the sheep were able to cope with these increases in stocking density under the conditions imposed, but the conclusions must be interpreted in the context of the controlled experimental conditions.

Corrigendum: Stocking density, restricted trough space, and implications for sheep behaviour and biological functioning.

[This corrects the article DOI: 10.3389/fvets.2022.965635.].

Stocking density, restricted trough space, and implications for sheep behaviour and biological functioning.

Stocking density and trough space allowance can potentially impact sheep welfare during live export voyages. The aim of this study was to assess the welfare implications for sheep housed at five allometric stocking densities, with either unrestricted or restricted trough space allowance. Merino wethers (n = 720) were housed in 40 pens of 18 heads for 18 days. Two 5-min continuous focal animal observations (n = 3/pen) were conducted on days 3, 5, 11, and 17. Scan sampling of standing and lying behaviours were conducted on the same days at hourly intervals. Live weights and immune cell counts were quantified at the start and end of the experiment, as well as faecal glucocorticoid metabolites (FGCMs), which were also assessed on days 6 and 12. Focal animals housed at higher stocking densities spent less time lying during one of the continuous observation periods, but no important effects on the overall number of animals lying or on the synchronicity of lying were evident. The scan sampling results indicated that the expression of some preferred lying positions was impaired at high stocking densities, and that high stocking densities also resulted in increased agonistic social interactions and displacement events at the start of the trial. There was a slight reduction in day 18 live weights for animals housed at higher stocking densities, but FGCM concentrations and immune cell counts were essentially unaffected. Trough space had no important effects on day 18 live weight, FGCM concentrations, or immune cell counts, and had limited effects on sheep behaviour. The lack of important impacts on biological fitness traits suggests that the behavioural responses observed were sufficient in allowing sheep to cope with their environment. However, we provide evidence that the provision of additional space is beneficial in reducing the time it takes for animals to adapt to their environment and to facilitate the expression of some preferred lying positions. While designed to emulate certain conditions relevant during live export voyages, some factors that may induce stress during this mode of transport were not present such as heat and ocean swell, so the conclusions must be interpreted in the context of the experimental conditions.

Point prevalence of non-melanoma and melanoma skin cancers in Australian surfers and swimmers in Southeast Queensland and Northern New South Wales.

Background: Surfing and swimming are two popular outdoor aquatic activities in Australia with an estimated 2.7 million surfers and three million swimmers; however, these activities are associated with intermittent exposure to ultraviolet radiation. Our aim was to determine the point prevalence of pre-skin cancer (actinic keratosis (PSC)), non-melanoma (NMSC) and melanoma skin cancers (MSC) in Australian surfers and swimmers. Methods: This cross-sectional study involved Australian surfers who completed a survey that included physiological demographics, aquatic activity-specific demographics, history of skin cancer followed by screening. Results: A total of 171 surfers (n = 116) and swimmers (n = 55) participated in the study. Both groups were identified as having a history of skin cancer (surfers 41.4%, swimmers 36.4%) and a family history of skin cancer (surfers 52.6%, swimmers 43.6%). The majority of both groups reported using a high percentage of a chemical or physical skin cancer prevention strategy (surfers 100%, Swimmers 92.7%, P = 0.003). Significantly more surfers were identified with a skin cancer of any type vs. swimmers (50% vs. 27.3%; OR 2.67; P = 0.005) with most the common skin cancer being PSC (44.7% vs. 11.3%, P = 0.076) followed by basal cell carcinoma (BCC) (24.2% vs. 7.6%, P = 0.068). There was a total of seven MSC identified in surfers and swimmers (4.6% vs. 0.8%, respectively, P = 0.137). Most skin cancers in surfers were located on the face (28.0%) followed by the arm and back (12.1% each), whereas in swimmers, the majority of skin cancers were identified on the face (17.3%), followed by the arm and lower leg (15.4% each). The highest number of melanomas were identified in surfers (n = 6) and mainly located on the face (n = 2) and back (n = 2). There was a single melanoma identified on the back in a swimmer. With the groups combined, the majority (42.9%) of melanomas were identified on the back in participants, followed by the face (28.6%). Rates per 100,000 of NMSC and MSC in surfers and swimmers (respectively) were BCC (11,206 vs. 14,545), squamous cell carcinoma (SCC) in situ (13,793 vs. 12,727), SCC (1,724 vs. 3,636) and MSC (5,172 vs. 1,818). When compared to the general Australian population, surfers and swimmers had higher odds ratios (OR), which included BCCs (OR 7.3 and 9.4, respectively), SCCs (OR 1.7 and 3.5, respectively) and MSC (OR 96.7 and 18.8, respectively). Conclusion: Surfers and swimmers had consistently higher rates of PSC, NMSC and MSC than the general Australian population. Point prevalence of MSC (groups combined) was 76-fold higher than the general Australian population. These findings highlight the clinical importance of regular skin cancer screenings in individuals who surf or swim for early detection and treatment of skin cancer. Additionally, these aquatic enthusiasts should be advised of the benefits of sun protection strategies such as chemical and physical barriers to reduce the likelihood of developing skin cancer.

The utility of a genetic kidney disease clinic employing a broad range of genomic testing platforms: experience of the Irish Kidney Gene Project.

BACKGROUND AND AIMS: Genetic testing presents a unique opportunity for diagnosis and management of genetic kidney diseases (GKD). Here, we describe the clinical utility and valuable impact of a specialized GKD clinic, which uses a variety of genomic sequencing strategies. METHODS: In this prospective cohort study, we undertook genetic testing in adults with suspected GKD according to prespecified criteria. Over 7 years, patients were referred from tertiary centres across Ireland to an academic medical centre as part of the Irish Kidney Gene Project. RESULTS: Among 677 patients, the mean age was of 37.2 ± 13 years, and 73.9% of the patients had family history of chronic kidney disease (CKD). We achieved a molecular diagnostic rate of 50.9%. Four genes accounted for more than 70% of identified pathogenic variants: PKD1 and PKD2 (n = 186, 53.4%), MUC1 (8.9%), and COL4A5 (8.3%). In 162 patients with a genetic diagnosis, excluding PKD1/PKD2, the a priori diagnosis was confirmed in 58% and in 13% the diagnosis was reclassified. A genetic diagnosis was established in 22 (29.7%) patients with CKD of uncertain aetiology. Based on genetic testing, a diagnostic kidney biopsy was unnecessary in 13 (8%) patients. Presence of family history of CKD and the underlying a priori diagnosis were independent predictors (P < 0.001) of a positive genetic diagnosis. CONCLUSIONS: A dedicated GKD clinic is a valuable resource, and its implementation of various genomic strategies has resulted in a direct, demonstrable clinical and therapeutic benefits to affected patients.

Use your head: overcoming epicardial complications during a single-catheter chronic total occlusion-a case-based review.

The success of attempts at opening chronic total occlusions (CTO) has dramatically increased in recent times due to the development of new techniques such as the use of the retrograde approach through epicardial collaterals. However, this approach admittedly brings with it an increased risk, and this must be balanced against the potential benefits. We present the case of a 61-year-old gentleman with Canadian Cardiovascular Society (CCS) Class III angina with a background history of hypertension and dyslipidaemia, who was an ex-smoker, and whose diagnostic coronary angiogram revealed CTOs of both the right and left circumflex coronary arteries. Following a heart team discussion, a percutaneous approach to treatment by staged approach was favoured, with the first stage being opening of the CTO of the right coronary artery. A retrograde approach with the use of a Corsair microcatheter facilitated reverse CART (controlled antegrade and retrograde tracking). Unfortunately, upon removal of the Corsair, a rupture of the epicardial collateral was noted with profuse bleeding into the pericardial space. This was treated successfully with a BeGraft-covered stent to obtain proximal control, and a Cooke Tornado neuro-interventional coil to obtain distal control, delivered antegrade through the now recanalized RCA. This case-based review then highlights several unique learning points, in particular to understand, in general terms, the approach to CTO; to understand the potential complications associated with a retrograde epicardial collateral approach; to understand the stepwise approach to dealing with perforation; and, finally, to understand how an occlusion coil works.

A Rare Autosomal Dominant Variant in Regulator of Calcineurin Type 1 (RCAN1) Gene Confers Enhanced Calcineurin Activity and May Cause FSGS.

BACKGROUND: Podocyte dysfunction is the main pathologic mechanism driving the development of FSGS and other morphologic types of steroid-resistant nephrotic syndrome (SRNS). Despite significant progress, the genetic causes of most cases of SRNS have yet to be identified. METHODS: Whole-genome sequencing was performed on 320 individuals from 201 families with familial and sporadic NS/FSGS with no pathogenic mutations in any known NS/FSGS genes. RESULTS: Two variants in the gene encoding regulator of calcineurin type 1 (RCAN1) segregate with disease in two families with autosomal dominant FSGS/SRNS. In vitro, loss of RCAN1 reduced human podocyte viability due to increased calcineurin activity. Cells expressing mutant RCAN1 displayed increased calcineurin activity and NFAT activation that resulted in increased susceptibility to apoptosis compared with wild-type RCAN1. Treatment with GSK-3 inhibitors ameliorated this elevated calcineurin activity, suggesting the mutation alters the balance of RCAN1 regulation by GSK-3ß, resulting in dysregulated calcineurin activity and apoptosis. CONCLUSIONS: These data suggest mutations in RCAN1 can cause autosomal dominant FSGS. Despite the widespread use of calcineurin inhibitors in the treatment of NS, genetic mutations in a direct regulator of calcineurin have not been implicated in the etiology of NS/FSGS before this report. The findings highlight the therapeutic potential of targeting RCAN1 regulatory molecules, such as GSK-3ß, in the treatment of FSGS.

Gene delivery to the hypoglossal motor system: preclinical studies and translational potential.

Dysfunction and/or reduced activity in the tongue muscles contributes to conditions such as dysphagia, dysarthria, and sleep disordered breathing. Current treatments are often inadequate, and the tongue is a readily accessible target for therapeutic gene delivery. In this regard, gene therapy specifically targeting the tongue motor system offers two general strategies for treating lingual disorders. First, correcting tongue myofiber and/or hypoglossal (XII) motoneuron pathology in genetic neuromuscular disorders may be readily achieved by intralingual delivery of viral vectors. The retrograde movement of viral vectors such as adeno-associated virus (AAV) enables targeted distribution to XII motoneurons via intralingual viral delivery. Second, conditions with impaired or reduced tongue muscle activation can potentially be treated using viral-driven chemo- or optogenetic approaches to activate or inhibit XII motoneurons and/or tongue myofibers. Further considerations that are highly relevant to lingual gene therapy include (1) the diversity of the motoneurons which control the tongue, (2) the patterns of XII nerve branching, and (3) the complexity of tongue muscle anatomy and biomechanics. Preclinical studies show considerable promise for lingual directed gene therapy in neuromuscular disease, but the potential of such approaches is largely untapped.

Integration of genetic and histopathology data in interpretation of kidney disease.

For many years renal biopsy has been the gold standard for diagnosis in many forms of kidney disease. It provides rapid, accurate and clinically useful information in most individuals with kidney disease. However, in recent years, other diagnostic modalities have become available that may provide more detailed and specific diagnostic information in addition to, or instead of, renal biopsy. Genomics is one of these modalities. Previously prohibitively expensive and time consuming, it is now increasingly available and practical in a clinical setting for the diagnosis of inherited kidney disease. Inherited kidney disease is a significant cause of kidney disease, in both the adult and paediatric populations. While individual inherited kidney diseases are rare, together they represent a significant burden of disease. Because of the heterogenicity of inherited kidney disease, diagnosis and management can be a challenge and often multiple diagnostic modalities are needed to arrive at a diagnosis. We present updates in genomic medicine for renal disease, how genetic testing integrates with our knowledge of renal histopathology and how the two modalities may interact to enhance patient care.

Diagnostic utility of genetic testing in patients undergoing renal biopsy.

High-throughput DNA testing is becoming established as a standard diagnostic test in the renal clinic. Previously published studies on cohorts of patients with unexplained chronic kidney disease of a suspected genetic aetiology have suggested a diagnostic yield for genomic sequencing of up to 18%. Here we determine the yield of targeted gene panel in a clinically unscreened cohort of patients referred for percutaneous native renal biopsy. Patients who underwent renal biopsy for investigation of chronic kidney disease were sequenced using a genomic sequencing panel covering 227 genes in which variation is known to be associated with monogenic chronic kidney disease (CKD). Candidate disease-causing variants were assessed for pathogenicity using guidelines from the American College for Medical Genetics and Genomics. Fifty CKD patients were recruited and sequenced. A molecular diagnosis was obtained for two patients (4%). A molecular diagnosis is possible using genomic testing in ∼4% of clinically unscreened patients undergoing renal biopsy. Genetic screening may be useful for diagnosis in a subset of CKD patients but is most valuable when applied to patients with suspected heritable forms of kidney disease.

Renal transplant outcomes in patients with autosomal dominant tubulointerstitial kidney disease.

INTRODUCTION: Autosomal dominant tubulointerstitial kidney disease (ADTKD) is a rare genetic cause of chronic kidney disease (CKD) and end-stage renal disease (ESRD). We aimed to compare renal transplant outcomes in people with ESRD due to ADTKD to those with other causes of renal failure. METHODS: Patients with clinical characteristics consistent with ADTKD by the criteria outlined in the 2015 KDIGO consensus were included. We compared ADTKD transplant outcomes with those of 4633 non-ADTKD renal transplant recipients. RESULTS: We included 31 patients who met diagnostic criteria for ADTKD in this analysis, 23 of whom had an identified mutation (28 were categorized as definite-ADTKD and 3 as suspected ADTKD). Five patients received a second transplant during follow-up. In total, 36 grafts were included. We did not identify significant differences between groups in terms of graft or patient survival after transplantation. Twenty-five transplant biopsies were performed during follow-up, and none of these showed signs of recurrent ADTKD post-transplant. CONCLUSION: In patients with ESRD due to ADTKD, we demonstrate that transplant outcomes are comparable with the general transplant population. There is no evidence that ADTKD can recur after transplantation.

Karyomegalic Interstitial Nephritis: Cancer Risk Following Transplantation.

A brother and sister presented individually in their forties with progressive renal failure, bronchiectasis and mild derangements of their liver function tests. Both developed end-stage renal disease before the age of 50. Both siblings were found to carry a pathogenic, recessive, compound heterozygote mutation in the gene FAN1, which causes karyomegalic interstitial nephritis. Both siblings underwent renal transplantation. The sister developed small cell carcinoma of the lung 18 months after transplantation. Despite intensive chemotherapy she died 6 months later. Six years after transplant, the brother has developed prostate cancer and over 30 individual skin cancers. To our knowledge, only 6 other patients with FAN1 mutations have undergone solid organ transplantation. Outcomes have been poor, with only 3 patients surviving beyond 1 year. While cancer is a significant risk for all patients in the post-transplant period, only 0.01% of patients develop >10 skin cancers. Transplantation may be an unrecognised risk in patients with FAN1 mutations.

Utility of Genomic Testing after Renal Biopsy.

BACKGROUND: Renal biopsy is the mainstay of renal pathological diagnosis. Despite sophisticated diagnostic techniques, it is not always possible to make a precise pathological diagnosis. Our aim was to identify a genetic cause of disease in patients who had undergone renal biopsy and determine if genetic testing altered diagnosis or treatment. METHODS: Patients with suspected familial kidney disease underwent a variety of next-generation sequencing (NGS) strategies. The subset of these patients who had also undergone native kidney biopsy was identified. Histological specimens were reviewed by a consultant pathologist, and genetic and pathological diagnoses were compared. RESULTS: Seventy-five patients in 47 families underwent genetic sequencing and renal biopsy. Patients were grouped into 5 diagnostic categories based on pathological diagnosis: tubulointerstitial kidney disease (TIKD; n = 18); glomerulonephritis (GN; n = 15); focal segmental glomerulosclerosis and Alport Syndrome (n = 11); thrombotic microangiopathy (TMA; n = 17); and nonspecific pathological changes (n = 14). Thirty-nine patients (52%) in 21 families (45%) received a genetic diagnosis; 13 cases (72%) with TIKD, 4 (27%) with GN, 6 (55%) with focal segmental glomerulosclerosis/Alport syndrome, and 10 (59%) with TMA and 6 cases (43%) with nonspecific features. Genetic testing resulted in changes in understanding of disease mechanism in 21 individuals (54%) in 12 families (57%). Treatment would have been altered in at least 26% of cases (10/39). CONCLUSIONS: An accurate genetic diagnosis can result in changes in clinical diagnosis, understanding of pathological mechanism, and treatment. NGS should be considered as a complementary diagnostic technique to kidney biopsy in the evaluation of patients with kidney disease.

An Exome Sequencing Study of 10 Families with IgA Nephropathy.

BACKGROUND: Immunoglobulin A nephropathy (IgAN) is a heterogeneous disorder with a strong genetic component. The advent of whole exome sequencing (WES) has accelerated the discovery of genetic risk factors underlying familial disorders. OBJECTIVES: We set out to test whether damaging variants in known kidney disease genes explain a proportion of IgAN cases recruited in Ireland. METHODS: We performed WES in 10 Irish families with multiple affected members having kidney disease where at least one member had biopsy confirmed IgAN. Candidate variants were identified based on being shared between affected family members, minor allele frequency, function and predicted pathogenicity. Pathogenicity of variants was determined according to American College of Medical Genetics and Genomics guidelines. RESULTS: We detected candidate variants in 3 of 10 families. We identified a likely pathogenic variant in COL4A5 in one family and a variant of unknown significance (VUS) in COL4A3 in another. Variants in COL4A5 and COL4A3 are known to cause Alport syndrome. In the third family, we identified a VUS in LMX1B, a gene associated with Nail-patella syndrome. CONCLUSIONS: We identified a number of cases of familial IgAN where the families harbored variants in known kidney disease-related genes indicating that potentially a number of cases of familial IgAN are mistaken for other familial kidney disorders. However, the majority of families studied did not carry a candidate variant in a known kidney disease causing gene indicating that there may be >1 underlying genetic mechanism present in these families.

AAV Gene Therapy Utilizing Glycosylation-Independent Lysosomal Targeting Tagged GAA in the Hypoglossal Motor System of Pompe Mice.

Pompe disease is caused by mutations in the gene encoding the lysosomal glycogen-metabolizing enzyme, acid-alpha glucosidase (GAA). Tongue myofibers and hypoglossal motoneurons appear to be particularly susceptible in Pompe disease. Here we used intramuscular delivery of adeno-associated virus serotype 9 (AAV9) for targeted delivery of an enhanced form of GAA to tongue myofibers and motoneurons in 6-month-old Pompe (Gaa -/- ) mice. We hypothesized that addition of a glycosylation-independent lysosomal targeting tag to the protein would result in enhanced expression in tongue (hypoglossal) motoneurons when compared to the untagged GAA. Mice received an injection into the base of the tongue with AAV9 encoding either the tagged or untagged enzyme; tissues were harvested 4 months later. Both AAV9 constructs effectively drove GAA expression in lingual myofibers and hypoglossal motoneurons. However, mice treated with the AAV9 construct encoding the modified GAA enzyme had a >200% increase in the number of GAA-positive motoneurons as compared to the untagged GAA (p < 0.008). Our results confirm that tongue delivery of AAV9-encoding GAA can effectively target tongue myofibers and associated motoneurons in Pompe mice and indicate that the effectiveness of this approach can be improved by addition of the glycosylation-independent lysosomal targeting tag.