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Noonan syndrome (NS) is the most common disease among RASopathies, characterized by short stature, distinctive facial features, congenital cardiac defects, and variable developmental delay. NS rarely presents with overt neurologic manifestations, in particular hydrocephalus. Recent evidence suggests that pathogenic variants in the gene RRAS2 are a rare cause of NS. Specifically, an RRAS2 pathogenic variant, p.Q72L, may be particularly severe, manifesting with lethal neurologic findings. Here, we report a NS patient with documented p.Q72L variant in RRAS2. The patient was identified in utero to have hydrocephalus and a Dandy Walker malformation. Postnatal examination revealed multiple dysmorphic features, some reminiscent of NS including low-set posteriorly rotated ears, redundant nuchal skin, widely spaced nipples, and cryptorchidism. Despite suspicion of NS, results of a 14-gene Noonan syndrome panel (Invitae) were negative. Follow-up rapid whole exome sequencing revealed a de novo p.Q72L variant in RRAS2, a poorly studied gene recently identified as a cause of NS. The patient herein reported brings to three the total number of cases reported with the RRAS2 p.Q72L pathogenic variant. All three documented patients presented with a particularly fulminant course of NS, which included hydrocephalus. RRAS2, specifically p.Q72L, should be considered in severe NS cases with neurologic manifestations.

Globoid cell leukodystrophy (GLD), also known as Krabbe disease, is a lysosomal storage disorder causing extensive demyelination in the central and peripheral nervous systems. GLD is caused by loss-of-function mutations in the lysosomal hydrolase, galactosylceramidase (GALC), which catabolizes the myelin sphingolipid galactosylceramide. The pathophysiology of GLD is complex and reflects the expression of GALC in a number of glial and neural cell types in both the central and peripheral nervous systems (CNS and PNS), as well as leukocytes and kidney in the periphery. Over the years, GLD has garnered a wide range of scientific and medical interests, especially as a model system to study gene therapy and novel preclinical therapeutic approaches to treat the spontaneous murine model for GLD. Here, we review recent findings in the field of Krabbe disease, with particular emphasis on novel aspects of GALC physiology, GLD pathophysiology, and therapeutic strategies.

Myelin is essential for rapid nerve impulse propagation and axon protection. Accordingly, defects in myelination or myelin maintenance lead to secondary axonal damage and subsequent degeneration. Studies utilizing genetic (CNPase-, MAG-, and PLP-null mice) and naturally occurring neuropathy models suggest that myelinating glia also support axons independently from myelin. Myelin protein zero (MPZ or P0), which is expressed only by Schwann cells, is critical for myelin formation and maintenance in the peripheral nervous system. Many mutations in MPZ are associated with demyelinating neuropathies (Charcot-Marie-Tooth disease type 1B [CMT1B]). Surprisingly, the substitution of threonine by methionine at position 124 of P0 (P0T124M) causes axonal neuropathy (CMT2J) with little to no myelin damage. This disease provides an excellent paradigm to understand how myelinating glia support axons independently from myelin. To study this, we generated targeted knock-in P0T124M mutant mice, a genetically authentic model of T124M-CMT2J neuropathy. Similar to patients, these mice develop axonopathy between 2 and 12 months of age, characterized by impaired motor performance, normal nerve conduction velocities but reduced compound motor action potential amplitudes, and axonal damage with only minor compact myelin modifications. Mechanistically, we detected metabolic changes that could lead to axonal degeneration, and prominent alterations in non-compact myelin domains such as paranodes, Schmidt-Lanterman incisures, and gap junctions, implicated in Schwann cell-axon communication and axonal metabolic support. Finally, we document perturbed mitochondrial size and distribution along P0T124M axons suggesting altered axonal transport. Our data suggest that Schwann cells in P0T124M mutant mice cannot provide axons with sufficient trophic support, leading to reduced ATP biosynthesis and axonopathy. In conclusion, the P0T124M mouse model faithfully reproduces the human neuropathy and represents a unique tool for identifying the molecular basis for glial support of axons.

Krabbe disease is caused by a deficiency of the lysosomal galactosylceramidase (GALC) enzyme, which results in the accumulation of galactosylceramide (GalCer) and psychosine. In Krabbe disease, the brunt of demyelination and neurodegeneration is believed to result from the dysfunction of myelinating glia. Recent studies have shown that neuronal axons are both structurally and functionally compromised in Krabbe disease, even before demyelination, suggesting a possible neuron-autonomous role of GALC. Using a novel neuron-specific Galc knockout (CKO) model, we show that neuronal Galc deletion is sufficient to cause growth and motor coordination defects and inflammatory gliosis in mice. Furthermore, psychosine accumulates significantly in the nervous system of neuron-specific Galc-CKO. Confocal and electron microscopic analyses show profound neuro-axonal degeneration with a mild effect on myelin structure. Thus, we prove for the first time that neuronal GALC is essential to maintain and protect neuronal function independently of myelin and may directly contribute to the pathogenesis of Krabbe disease.

Krabbe disease (KD) is caused by a deficiency of galactosylceramidase (GALC), which induces demyelination and neurodegeneration due to accumulation of cytotoxic psychosine. Hematopoietic stem cell transplantation (HSCT) improves clinical outcomes in KD patients only if delivered pre-symptomatically. Here, we hypothesize that the restricted temporal efficacy of HSCT reflects a requirement for GALC in early brain development. Using a novel Galc floxed allele, we induce ubiquitous GALC ablation (Galc-iKO) at various postnatal timepoints and identify a critical period of vulnerability to GALC ablation between P4-6 in mice. Early Galc-iKO induction causes a worse KD phenotype, higher psychosine levels in the rodent brainstem and spinal cord, and a significantly shorter life-span of the mice. Intriguingly, GALC expression peaks during this critical developmental period in mice. Further analysis of this mouse model reveals a cell autonomous role for GALC in the development and maturation of immature T-box-brain-1 positive brainstem neurons. These data identify a perinatal developmental period, in which neuronal GALC expression influences brainstem development that is critical for KD pathogenesis., Krabbe disease is caused by GALC deficiency, leading to accumulation of cytotoxic psychosine, demyelination, and neurodegeneration. Here, the authors develop a Galc flox mouse line to model Krabbe disease and unveil that early postnatal GALC neuronal expression is critical for disease pathogenesis.

Summary Many therapies for lysosomal storage disorders rely on cross-correction of lysosomal enzymes. In globoid cell leukodystrophy (GLD), mutations in GALC cause psychosine accumulation, inducing demyelination, a neuroinflammatory “globoid” reaction and neurodegeneration. The efficiency of GALC cross-correction in vivo, the role of the GALC substrate galactosylceramide, and the origin of psychosine are poorly understood. Using a novel GLD model, we show that cross-correction does not occur efficiently in vivo and that Galc-deficient Schwann cells autonomously produce psychosine. Furthermore, macrophages require GALC to degrade myelin, as Galc-deficient macrophages are transformed into globoid cells by exposure to galactosylceramide and produce a more severe GLD phenotype. Finally, hematopoietic stem cell transplantation in patients reduces globoid cells in nerves, suggesting that the phagocytic response of healthy macrophages, rather than cross-correction, contributes to the therapeutic effect. Thus, GLD may be caused by at least two mechanisms: psychosine-induced demyelination and secondary neuroinflammation from galactosylceramide storage in macrophages.

There are over 50 lysosomal hydrolase deficiencies, many of which cause neurodegeneration, cognitive decline and death. In recent years, a number of broad innovative therapies have been proposed and investigated for lysosomal storage diseases (LSDs), such as enzyme replacement, substrate reduction, pharmacologic chaperones, stem cell transplantation, and various forms of gene therapy. Murine models that accurately reflect the phenotypes observed in human LSDs are critical for the development, assessment and implementation of novel translational therapies. The goal of this review is to summarize the neurodegenerative murine LSD models available that recapitulate human disease, and the pre-clinical studies previously conducted. We also describe some limitations and difficulties in working with mouse models of neurodegenerative LSDs.

Krabbe disease (KD) is caused by a deficiency of galactosylceramidase (GALC), which induces demyelination and neurodegeneration due to accumulation of cytotoxic psychosine. Hematopoietic stem cell transplantation (HSCT) improves clinical outcomes in KD patients only if delivered pre-symptomatically. We hypothesized that the restricted temporal efficacy of HSCT reflects a requirement for GALC in early brain development. Using a novel Galc floxed allele, we induced ubiquitous GALC ablation (Galc-iKO) at various postnatal timepoints and identified a critical period of vulnerability to GALC ablation between P4-6. Early Galc-iKO induction caused a worse KD phenotype, higher psychosine levels, and a significantly shorter life-span. Intriguingly, GALC expression peaks during this critical developmental period. Further analysis revealed a novel cell autonomous role for GALC in the development and maturation of immature T-box-brain-1 positive brainstem neurons. These data identify a perinatal developmental period, in which neuronal GALC expression influences brainstem development that is critical for KD pathogenesis.

Many therapies for lysosomal storage disorders relies on cross-correction of lysosomal enzymes. In globoid cell leukodystrophy (GLD), mutations in GALC cause psychosine accumulation, inducing demyelination, a neuroinflammatory “globoid” reaction and neurodegeneration. The efficiency of GALC cross-correction in vivo , the role of the GALC substrate galactosylceramide, and the origin of psychosine are poorly understood. Using a novel GLD model, we show that cross-correction does not occur efficiently in vivo , and that GALC-deficient Schwann cells autonomously produce psychosine. Furthermore, macrophages require GALC to degrade myelin, and GALC-deficient macrophages are transformed into globoid cells by exposure to galactosylceramide and consequently produce a more severe GLD phenotype. Finally, hematopoietic stem cell transplantation in patients reduces globoid cells in nerves, suggesting that the phagocytic response of healthy macrophages, rather than cross-correction, contributes to the therapeutic effect. Thus, GLD may be caused by at least two mechanisms: psychosine-induced demyelination and secondary neuroinflammation from galactosylceramide storage in macrophages.

Krabbe disease (KD) is caused by mutations in the galactosylceramidase (GALC) gene, which encodes a lysosomal enzyme that degrades galactolipids, including galactosylceramide and galactosylsphingosine (psychosine). GALC deficiency results in progressive intracellular accumulation of psychosine, which is believed to be the main cause for the demyelinating neurodegeneration in KD pathology. Umbilical cord blood transplantation slows disease progression when performed presymptomatically but carries a significant risk of morbidity and mortality. Accurate presymptomatic diagnosis is therefore critical to facilitate the efficacy of existing transplant approaches and to avoid unnecessary treatment of children who will not develop KD. Unfortunately, current diagnostic criteria, including GALC activity, genetic analysis, and psychosine measurement, are insufficient for secure presymptomatic diagnosis. This study performs a global metabolomic analysis to identify pathogenetic metabolic pathways and novel biomarkers implicated in the authentic mouse model of KD known as twitcher. At a time point before onset of signs of disease, twitcher hindbrains had metabolic profiles similar to WT, with the exception of a decrease in metabolites related to glucose energy metabolism. Many metabolic pathways were altered after early signs of disease in the twitcher, including decreased phospholipid turnover, restricted mitochondrial metabolism of branched-chain amino acids, increased inflammation, and changes in neurotransmitter metabolism and osmolytes. Hypoxanthine, a purine derivative, is increased before signs of disease appear, suggesting its potential as a biomarker for early diagnosis of KD. Additionally, given the early changes in glucose metabolism in the pathogenesis of KD, diagnostic modalities that report metabolic function, such as positron emission tomography, may be useful in KD. © 2016 Wiley Periodicals, Inc.

Smith-Lemli-Opitz Syndrome (SLOS) is a recessive human disease caused by defective cholesterol (CHOL) synthesis at the level of DHCR7 (7-dehydrocholesterol reductase), which normally catalyzes the conversion of 7-dehydrocholesterol (7DHC) to CHOL. Formation and abnormal accumulation of 7DHC and 7DHC-derived oxysterols occur in SLOS patients and in rats treated with the DHCR7 inhibitor AY9944. The rat SLOS model exhibits progressive and irreversible retinal dysfunction and degeneration, which is only partially ameliorated by dietary CHOL supplementation. We hypothesized that 7DHC-derived oxysterols are causally involved in this retinal degeneration, and that blocking or reducing their formation should minimize the phenotype. Here, using the SLOS rat model, we demonstrate that combined dietary supplementation with CHOL plus antioxidants (vitamins E and C, plus sodium selenite) provides better outcomes than dietary CHOL supplementation alone with regard to preservation of retinal structure and function and lowering 7DHC-derived oxysterol formation. These proof-of-principle findings provide a translational, pre-clinical framework for designing clinical trials using CHOL-antioxidant combination therapy as an improved therapeutic intervention over the current standard of care for the treatment of SLOS.

The dorsal cochlear nucleus (DCN) is a brainstem structure that receives input from the auditory nerve. Many studies in a diversity of species have shown that the DCN has a laminar organization and identifiable neuron types with predictable synaptic relations to each other. In contrast, studies on the human DCN have found a less distinct laminar organization and fewer cell types, although there has been disagreement among studies in how to characterize laminar organization and which of the cell types identified in other animals are also present in humans. We have reexamined DCN organization in the human using immunohistochemistry to analyze the expression of several proteins that have been useful in delineating the neurochemical organization of other brainstem structures in humans: nonphosphorylated neurofilament protein (NPNFP), nitric oxide synthase (nNOS), and three calcium-binding proteins. The results for humans suggest a laminar organization with only two layers, and the presence of large projection neurons that are enriched in NPNFP. We did not observe evidence in humans of the inhibitory interneurons that have been described in the cat and rodent DCN. To compare humans and other animals directly we used immunohistochemistry to examine the DCN in the macaque monkey, the cat, and three rodents. We found similarities between macaque monkey and human in the expression of NPNFP and nNOS, and unexpected differences among species in the patterns of expression of the calcium-binding proteins.

Krabbe disease (KD) is caused by mutations in the galactosylceramidase (GALC) gene, which encodes a lysosomal enzyme that degrades galactolipids, including galactosylceramide and galactosylsphingosine (psychosine). GALC deficiency results in progressive intracellular accumulation of psychosine, which is believed to be the main cause for the demyelinating neurodegeneration in KD pathology. Umbilical cord blood transplantation slows disease progression when performed presymptomatically but carries a significant risk of morbidity and mortality. Accurate presymptomatic diagnosis is therefore critical to facilitate the efficacy of existing transplant approaches and to avoid unnecessary treatment of children who will not develop KD. Unfortunately, current diagnostic criteria, including GALC activity, genetic analysis, and psychosine measurement, are insufficient for secure presymptomatic diagnosis. This study performs a global metabolomic analysis to identify pathogenetic metabolic pathways and novel biomarkers implicated in the authentic mouse model of KD known as twitcher. At a time point before onset of signs of disease, twitcher hindbrains had metabolic profiles similar to WT, with the exception of a decrease in metabolites related to glucose energy metabolism. Many metabolic pathways were altered after early signs of disease in the twitcher, including decreased phospholipid turnover, restricted mitochondrial metabolism of branched-chain amino acids, increased inflammation, and changes in neurotransmitter metabolism and osmolytes. Hypoxanthine, a purine derivative, is increased before signs of disease appear, suggesting its potential as a biomarker for early diagnosis of KD. Additionally, given the early changes in glucose metabolism in the pathogenesis of KD, diagnostic modalities that report metabolic function, such as positron emission tomography, may be useful in KD. © 2016 Wiley Periodicals, Inc.

The human cerebral cortex and cerebellum are greatly expanded compared to those of other mammals, including the great apes. This expansion is reflected in differences in the size and organization of precerebellar brainstem structures, such as the inferior olive. In addition, there are cell groups unique to the human brainstem. One such group may be the nucleus pararaphales (PRa); however, there is disagreement among authors about the size and location of this nucleus in the human brainstem. The name "pararaphales" has also been used for neurons in the medulla shown to project to the flocculus in the macaque monkey. We have re-examined the existence and status of the PRa in eight humans, three chimpanzees, and four macaque monkeys using Nissl-stained sections as well as immunohistochemistry. In the human we found a cell group along the midline of the medulla in all cases; it had the form of interrupted cell columns and was variable among cases in rostrocaudal and dorsoventral extent. Cells and processes were highly immunoreactive for non-phosphorylated neurofilament protein (NPNFP); somata were immunoreactive to the synthetic enzyme for nitric oxide, nitric oxide synthase, and for calretinin. In macaque monkey, there was a much smaller oval cell group with NPNFP immunoreactivity. In the chimpanzee, we found a region of NPNFP-immunoreactive cells and fibers similar to what was observed in macaques. These results suggest that the "PRa" in the human may not be the same structure as the flocculus-projecting cell group described in the macaque. The PRa, like the arcuate nucleus, therefore may be unique to humans.

Tinnitus, the perception of a phantom sound, is a common consequence of damage to the auditory periphery. A major goal of tinnitus research is to find the loci of the neural changes that underlie the disorder. Crucial to this endeavor has been the development of an animal behavioral model of tinnitus, so that neural changes can be correlated with behavioral evidence of tinnitus. Three major lines of evidence implicate the dorsal cochlear nucleus (DCN) in tinnitus. First, elevated spontaneous activity in the DCN is correlated with peripheral damage and tinnitus. Second, there are somatosensory inputs to the DCN that can modulate spontaneous activity and might mediate the somatic-auditory interactions seen in tinnitus patients. Third, we have found a subpopulation of DCN neurons in the adult rat that express doublecortin, a plasticity-related protein. The expression of this protein may reflect a role of these neurons in the neural reorganization causing tinnitus. However, there is a problem in extending the findings in the rodent DCN to humans. Classic studies state that the structure of the primate DCN is quite different from that of rodents, with primates lacking granule cells, the recipients of somatosensory input. To address the possibility of major species differences in DCN organization, we compared Nissl-stained sections of the DCN in five different species. In contrast to earlier reports, our data suggest that the organization of the primate DCN is not dramatically different from that of the rodents, and validate the use of animal data in the study of tinnitus. This article is part of a Special Issue entitled: Tinnitus Neuroscience.