Prion-like α-synuclein pathology in the brains of infants: Krabbe disease as a novel seed-competent α-synucleinopathy

Krabbe disease (KD) is an infantile neurodegenerative disorder resulting from pathogenic variants in the GALC gene which causes accumulation of the toxic sphingolipid psychosine. GALC variants are associated with increased risk of Lewy body diseases (LBD), an umbrella term for age-associated neurodegenerative diseases in which the protein α-synuclein aggregates into Lewy bodies. To explore whether α-synuclein in KD has pathological similarities to that in LBD, we compared post-mortem KD tissue to that of infant control cases and identified alterations to α-synuclein localisation and expression of modifications associated with LBD. To determine whether α-synuclein in KD displayed pathogenic properties associated with LBD we evaluated its seeding capacity using the real-time quaking-induced conversion assay. Strikingly, seeded aggregation of α-synuclein resulted in the formation of fibrillar aggregates similar to those observed in LBD, confirming the prion-like capacity of KD-derived α-synuclein. These observations constitute the first report of prion-like α-synuclein in the brain tissue of infants and challenge the putative view that α-synuclein pathology is merely an age-associated phenomenon, instead suggesting it can result from alterations to biological processes such as sphingolipid homeostasis. Our findings have important implications for understanding the mechanisms underlying Lewy body formation in LBD.

Advances in the Diagnosis and Treatment of Krabbe Disease

Krabbe disease is an autosomal recessive leukodystrophy caused by pathogenic variants in the galactocerebrosidase (GALC) gene. GALC activity is needed for the lysosomal hydrolysis of galactosylceramide, an important component of myelin. While most patients are infants, older patients are also diagnosed. Starting in 1970, a diagnosis could be made by measuring GALC activity in leukocytes and cultured cells. After the purification of GALC in 1993, the cDNA and genes were cloned. Over 260 disease-causing variants as well as activity lowering benign variants have been identified. While some pathogenic variants can be considered “severe,” others can be considered “mild.” The combination of alleles determines the type of Krabbe disease a person will have. To identify patients earlier, newborn screening (NBS) has been implemented in several states. Low GALC activity in this screening test may indicate a diagnosis of Krabbe disease. Second tier testing as well as neuro-diagnostic studies may be required to identify those individuals needing immediate treatment. Treatment of pre-symptomatic or mildly symptomatic patients at this time is limited to hematopoietic stem cell transplantation. Treatment studies using the mouse and dog models have shown that combining bone marrow transplantation with intra-venous gene therapy provides the best outcomes in terms of survival, behavior, and preservation of normal myelination in the central and peripheral nervous systems. With earlier diagnosis of patients through newborn screening and advances in treatment, it is hoped that more patients will have a much better quality of life.

Krabbe’s Disease mutation spectrum in Russian Federation

Болезнь Краббе (БКр) - редкое наследственное аутосомно-рецессивное заболевание, входящее в группу лизосомных болезней накопления. Заболевание обусловлено мутациями в гене GALC, приводящими к дефициту фермента галактозилцерамидазы. Частота БКр оценивается как 1:100 000 живых новорожденных, хотя в некоторых странах регистрируется более высокая частота заболевания. Точная частота БКр в Российской Федерации и в ее регионах неизвестна. Мажорной мутацией, приводящей к развитию БКр, является крупная делеция затрагивающая 11-17 экзоны гена GALC c.1161+6532_polyA+9kbdel (IVS10del30kb). Доля этой мутации в европейской популяции оценивается примерно в 50% от всех мутаций. Для изучения спектра и частот мутаций гена GALC на территории РФ были обследованы пациенты из разных регионов. Частая делеция составила 54% от общего числа выявленных мутаций, что сопоставимо с данными по европейской популяции. Однако в Чеченской Республике данная мутация встречалась гораздо чаще, чем в других регионах. Среди 950 исследованных образцов было выявлено 7 гетерозиготных носителей частой мутации. Учитывая вклад других мутаций, расчётная частота БКр в республике составила 1:51237, что превышает таковую в европейской популяции. Дополнительный анализ всех найденных мутаций гена GALC позволил выявить вариант c.578T>C, p.Ile193Thr, аллельная частота которого составила 8%. Данная замена впервые была описана у пациентов из нашей выборки и встречается только при поздней инфантильной форме заболевания у русских пациентов. Krabbe’s disease (KD) is a rare inherited autosomal recessive lysosomal storage disease. KD is caused by mutations in the GALC gene leading to deficiency of galactosylceramidase. KD oссurs in 1 per 100 000 newborns, although some countries have a higher incidence rate. The exact KD incidence in Russia is unknown. A major mutation leading to the KD development is a large deletion affecting exons 11-17 of the GALC gene с.1161+6532_polyA+9kbdel (IVS10del30kb). This mutation occurs in 50% of KD cases in the European population. Patients from different regions were studied to analyze the mutation spectrum and the incidence in the Russian population. The incidence rate of the large deletion in our study equals 54%, that is comparable with European population. However, in the Chechen Republic this mutation is much more common than in other regions. 950 samples were studied, 7 heterozygous carriers of frequent mutation were identified. Thus, the estimated KD incidence rate is 1:51237 considering other mutations, and it is higher than that in the European population. Additional analysis of all detected GALC mutations revealed a genetic variant c.578T>C, p.Ile193Thr with allelic frequency measured up 8%. This substitution was described in our selection for the first time and presented only in Russian patients with late infantile form of the disease.

Newborn Screening for Krabbe Disease—Illinois Experience: Role of Psychosine in Diagnosis of the Disease

Population-based newborn screening for Krabbe disease was initiated by measurement of galactocerebrosidase (GALC) activity in the state of Illinois in December 2017. Due to the poor specificity of GALC for the diagnosis of Krabbe disease, second-tier testing services were provided to reduce the false positive rates for disease monitoring. Using ultra-pressure liquid chromatography coupled to mass spectrometry assay, a total of 497,147 newborns were screened. In total, 288 infants’ specimens (0.06%) having reduced GALC activity were sent out for second-tier testing to a reference laboratory. All newborns’ reduced GALC specimens were tested for psychosine levels, the presence of a 30-kb deletion and GALC sequencing. The results showed that two infants had elevated psychosine levels (10 and 35 nM) and were referred immediately for evaluation and treatment for Infantile Krabbe disease, and six infants had intermediate PSY levels (≥2 to 5 nM) and are under observation as suspected candidates for late-onset Krabbe disease. In addition, 178 infants had pseudodeficiency alleles, all having psychosine levels < 2.0 nM. Our data show that a high percentage of reduced GALC activity (62%) was due to the presence of pseudodeficiency alleles in the GALC gene. In conclusion, incorporation of psychosine measurements can identify infants with infantile Krabbe disease and probable late-onset Krabbe infants. Furthermore, Krabbe disease screening can be achieved at public health laboratories, and infants with infantile Krabbe disease can be diagnosed in timely manner for better outcome.

Can early treatment of twitcher mice with high dose AAVrh10-GALC eliminate the need for BMT?

Introduction: Krabbe disease (KD) is an autosomal recessive disorder caused by mutations in the galactocerebrosidase (GALC) gene resulting in neuro-inflammation and defective myelination in the central and peripheral nervous systems. Most infantile patients present with clinical features before six months of age and die before two years of age. The only treatment available for pre-symptomatic or mildly affected individuals is hematopoietic stem cell transplantation (HSCT). In the animal models, combining bone marrow transplantation (BMT) with gene therapy has shown the best results in disease outcome. In this study, we examine the outcome of gene therapy alone. Methods: Twitcher (twi) mice used in the study, have a W339X mutation in the GALC gene. Genotype identification of the mice was performed shortly after birth or post-natal day 1 (PND1), using polymerase chain reaction on the toe clips followed by restriction enzyme digestion and electrophoresis. Eight or nine-day-old affected mice were used for gene therapy treatment alone or combined with BMT. While iv injection of 4 × 1013 gc/kg of body weight of viral vector was used originally, different viral titers were also used without BMT to evaluate their outcomes. Results: When the standard viral dose was increased four- and ten-fold (4X and 10X) without BMT, the lifespans were increased significantly. Without BMT the affected mice were fertile, had the same weight and appearance as wild type mice and had normal strength and gait. The brains showed no staining for CD68, a marker for activated microglia/macrophages, and less astrogliosis than untreated twi mice. Conclusion: Our results demonstrate that, it may be possible to treat human KD patients with high dose AAVrh10 without blood stem cell transplantation which would eliminate the side effects of HSCT.

AAV-Mediated GALC Gene Therapy Rescues Alpha-Synucleinopathy in the Spinal Cord of a Leukodystrophic Lysosomal Storage Disease Mouse Model

Krabbe's disease (KD) is primarily a demyelinating disorder, but recent studies have identified the presence of neuronal protein aggregates in the brain, at least partially composed by alpha-synuclein (α-syn). The role of this protein aggregation in the pathogenesis of KD is largely unknown, but it has added KD to a growing list of lysosomal storage diseases that can be also be considered as proteinopathies. While the presence of these protein aggregates within the KD brain is now appreciated, the remainder of the central nervous system (CNS) remains uncharacterized. This study is the first to report the presence of thioflavin-S reactive inclusions throughout the spinal cord of both murine and human spinal tissue. Stereological analysis revealed the temporal and spatial accumulation of these inclusions within the neurons of the ventral spinal cord vs. those located in the dorsal cord. This study also confirmed that these thio-S positive accumulations are present within neuronal populations and are made up at least in part by α-syn in both the twitcher mouse and cord autopsied material from affected human patients. Significantly, neonatal gene therapy for galactosylceramidase, a treatment that strongly improves the survival and health of KD mice, but not bone marrow transplantation prevents the formation of these inclusions in spinal neurons. These results expand the understanding of α-syn protein aggregation within the CNS of individuals afflicted with KD and underlines the tractability of this problem via early gene therapy, with potential impact to other synucleinopathies such as PD.