Assembly of the C1d projection of the central microtubule pair apparatus in mammalian motile cilia requires the ciliary protein CFAP54. Loss of the C1d projection in mice lacking CFAP54 impairs ciliary motility and cilia-driven fluid flow and results in a primary ciliary dyskinesia phenotype.

The juvenile onset form of neuronal ceroid lipofuscinoses (JNCL) is a recessively inherited lysosomal storage disorder characterized by progressive neurodegeneration. JNCL results from mutations in the CLN3 gene that encodes a lysosomal membrane protein with unknown function. Utilizing a Cln3-knock-out mouse model of JNCL that was created on the 129S6/SvEv genetic background, we have previously demonstrated that CLN3-deficient cerebellar granule cells (CGCs) have a selectively increased sensitivity to AMPA-type glutamate receptor-mediated toxicity. Our recent findings that CGCs from 129S6/SvEv and C57BL/6J wild type (WT) mice have significant differences in glutamate receptor expression and in excitotoxic vulnerability indicated that the genetic background possibly have a strong influence on how glutamate receptor function is dysregulated in CLN3-deficient neurons. Indeed, here we show that in the Cln3Δex7/8-knock-in mouse model, that is on the C57BL/6J genetic background, mimics the most frequent mutation observed in JNCL patients and considered a null mutant, the sensitivity of CGCs to both AMPA- and NMDA-type glutamate receptor overactivations is altered. Cultured wild type and Cln3Δex7/8 CGCs were equally sensitive to AMPA toxicity after 2 or 3 weeks in vitro, whereas the subunit-selective AMPA receptor agonist, CPW-399, induced significantly more cell death in mature, 3-week-old Cln3Δex7/8 cultures. NMDA receptor-mediated toxicity changed during in vitro development: Cln3Δex7/8 CGCs were less sensitive to high concentration of NMDA after 2 weeks in culture but became more vulnerable than their WT counterparts after 3 weeks in vitro. Abnormally altered glutamate receptor function in the cerebellum may result in motor deficits, and we confirmed that 7-week-old Cln3Δex7/8 mice, similarly to Cln3-knock-out mice, have a motor coordination deficit as measured by an accelerating rotarod. Our results demonstrate altered glutamate receptor function in Cln3Δex7/8 neurons and suggest that both AMPA and NMDA receptors are potential therapeutic targets in JNCL.

Hydrocephalus is caused by the accumulation of cerebrospinal fluid in the cerebral ventricular system which results in an enlargement of the cranium due to increased intraventricular pressure. The increase in pressure within the brain typically results in sloughing of ciliated ependymal cells, loss of cortical grey matter, and increased gliosis. Congenital hydrocephalus is associated with several syndromes including primary ciliary dyskinesia (PCD), a rare, genetically heterogeneous, pediatric syndrome that results from defects in motile cilia and flagella. We have examined the morphological and physiological defects in the brains of two mouse models of PCD, nm1054 and bgh, which have mutations in Pcdp1 (also known as Cfap221) and Spef2, respectively. Histopathological and immunohistochemical analyses of mice with these mutations on the C57BL/6J and 129S6/SvEvTac genetic backgrounds demonstrate strain-dependent morphological brain damage. Alterations in astrocytosis, microglial activation, myelination, and the neuronal population were identified and are generally more severe on the C57BL/6J background. Analysis of ependymal ciliary clearance ex vivo and cerebrospinal fluid flow in vivo demonstrate a physiological defect in nm1054 and bgh mice on both genetic backgrounds, indicating that abnormal cilia-driven flow is not the sole determinant of the severity of hydrocephalus in these models. These results suggest that genetic modifiers play an important role in susceptibility to severe PCD-associated hydrocephalus.

Alterations in glutamatergic synapse function have been implicated in the pathogenesis of many different neurological disorders including ischemia, epilepsy, Parkinson’s disease, Alzheimer’s disease, and Huntington’s disease. While studying glutamate receptor function in juvenile Batten disease on the C57BL/6J and 129S6/SvEv mouse backgrounds, we noticed differences unlikely to be due to mutation difference alone. We report here that primary cerebellar granule cell cultures from C57BL/6J mice are more sensitive to NMDA-mediated cell death. Moreover, sensitivity to AMPA-mediated excitotoxicity is more variable and is dependent upon the treatment conditions and age of the cultures. Glutamate receptor surface expression levels examined in vitro by in situ ELISA and in vivo by Western blot in surface cross-linked cerebellar samples indicated that these differences in sensitivity are likely due to strain-dependent differences in cell surface receptor expression levels. We propose that differences in glutamate receptor expression and in excitotoxic vulnerability should be taken into consideration in the context of characterizing disease models on the C57BL/6J and 129S6/SvEv mouse backgrounds.

The neuronal ceroid lipofuscinoses (NCLs) are a family of devastating pediatric neurodegenerative disorders and currently represent the most common form of pediatric-onset neurodegeneration. Infantile NCL (INCL), the most aggressive of these disorders, is caused by mutations in the CLN1 gene that encodes the enzyme palmitoyl protein thioesterase 1 (PPT1). Previous studies have suggested that glutamatergic neurotransmission may be disrupted in INCL, and therefore, the present study investigates glutamate receptor function in the Ppt1−/− mouse model of INCL by comparing the sensitivity of cultured WT and Ppt1−/− cerebellar granule cells to glutamate receptor-mediated toxicity. Ppt1−/− neurons were significantly less sensitive to AMPA receptor-mediated toxicity but markedly more vulnerable to NMDA receptor-mediated cell death. Since glutamate receptor function is primarily regulated by the surface expression level of the receptor, the surface level of AMPA and NMDA receptor subunits in the cerebella of WT and Ppt1−/− mice was also examined. Western blotting of surface cross-linked cerebellar samples showed a significantly lower surface level of the GluR4 AMPA receptor subunit in Ppt1−/− mice, providing a plausible explanation for the decreased vulnerability of Ppt1−/− cerebellar neurons to AMPA receptor-mediated cell death. The surface expression of the NR1, NR2A and NR2B NMDA receptor subunits was similar in the cerebella of WT and Ppt1−/− mice, indicating that there is another mechanism behind the increased sensitivity of Ppt1−/− cerebellar granule cells to NMDA toxicity. Our results indicate an AMPA receptor hypo- and NMDA receptor hyperfunction phenotype in Ppt1−/− neurons and provide new therapeutic targets for INCL.

Congenital hydrocephalus results from cerebrospinal fluid accumulation in the ventricles of the brain and causes severe neurological damage, but the underlying causes are not well understood. It is associated with several syndromes, including primary ciliary dyskinesia (PCD), which is caused by dysfunction of motile cilia. We previously demonstrated that mouse models of PCD lacking ciliary proteins CFAP221, CFAP54 and SPEF2 all have hydrocephalus with a strain-dependent severity. While morphological defects are more severe on the C57BL/6J (B6) background than 129S6/SvEvTac (129), cerebrospinal fluid flow is perturbed on both backgrounds, suggesting that abnormal cilia-driven flow is not the only factor underlying the hydrocephalus phenotype. Here, we performed a microarray analysis on brains from wild type and nm1054 mice lacking CFAP221 on the B6 and 129 backgrounds. Expression differences were observed for a number of genes that cluster into distinct groups based on expression pattern and biological function, many of them implicated in cellular and biochemical processes essential for proper brain development. These include genes known to be functionally relevant to congenital hydrocephalus, as well as formation and function of both motile and sensory cilia. Identification of these genes provides important clues to mechanisms underlying congenital hydrocephalus severity.

The neuronal ceroid lipofuscinoses, a family of neurodegenerative lysosomal storage disorders, represent the most common cause of pediatric-onset neurodegeneration. The infantile form has a devastatingly early onset and one of the fastest progressing disease courses. Despite decades of research, the molecular mechanisms driving neuronal loss in infantile neuronal ceroid lipofuscinosis remain unknown. We have previously shown that NMDA-type glutamate receptors in the Ppt1−/− mouse model of this disease exhibit a hyperfunctional phenotype and postulate that aberrant glutamatergic activity may contribute to neural pathology in both the mouse model and human patients. To test this hypothesis, we treated Ppt1−/− mice with the NMDA receptor antagonist memantine and assessed their response to the drug using an accelerating rotarod. At 20 mg/kg, memantine treatment induced a delayed but notable improvement in Ppt1−/− mice. Much remains to be assessed before moving to patient trials, but these results suggest memantine has potential as a treatment.

Patients with primary ciliary dyskinesia (PCD) suffer from a variety of disorders that include chronic sinusitis, otitis media, infertility, situs inversus, and hydrocephalus. We have shown that two mouse models of PCD, nm1054 and bgh, have hydrocephalus characterized by dilatation of the lateral ventricles. These models also have male infertility and sinus abnormalities that result from a decrease in respiratory ciliary beat frequency. We therefore hypothesize that the hydrocephalus results from dysfunction of ependymal cilia that prevents proper cerebrospinal fluid (CSF) flow. To investigate CSF flow in vivo, we injected India ink into the lateral ventricles of live mice and histologically demonstrated that ink migrated through the ventricular system of wild type mice but failed to progress past the cerebral aqueduct of either mutant. To validate that this defect was directly due to ciliary dysfunction, we used an ex vivo method to measure ink flow rate over the ependymal cells on dissected ventricular walls. This method demonstrated a decreased flow rate and altered directional flow in the mutant brains, confirming that ependymal ciliary function is required for proper CSF flow. Grant Funding Source: Supported by the NIH NIGMS (1P20GM103620‐01A1) and an NSF REU (1262744)