Microglial reduction of colony stimulating factor-1 receptor expression is sufficient to confer adult onset leukodystrophy.

Adult onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP) is a dementia resulting from dominantly inherited CSF1R inactivating mutations. The Csf1r+/- mouse mimics ALSP symptoms and pathology. Csf1r is mainly expressed in microglia, but also in cortical layer V neurons that are gradually lost in Csf1r+/- mice with age. We therefore examined whether microglial or neuronal Csf1r loss caused neurodegeneration in Csf1r+/- mice. The behavioral deficits, pathologies and elevation of Csf2 expression contributing to disease, previously described in the Csf1r+/- ALSP mouse, were reproduced by microglial deletion (MCsf1rhet mice), but not by neural deletion. Furthermore, increased Csf2 expression by callosal astrocytes, oligodendrocytes, and microglia was observed in Csf1r+/- mice and, in MCsf1rhet mice, the densities of these three cell types were increased in supraventricular patches displaying activated microglia, an early site of disease pathology. These data confirm that ALSP is a primary microgliopathy and inform future therapeutic and experimental approaches.

Loss-of-Huntingtin in Medial and Lateral Ganglionic Lineages Differentially Disrupts Regional Interneuron and Projection Neuron Subtypes and Promotes Huntington's Disease-Associated Behavioral, Cellular, and Pathological Hallmarks.

Emerging studies are providing compelling evidence that the pathogenesis of Huntington's disease (HD), a neurodegenerative disorder with frequent midlife onset, encompasses developmental components. Moreover, our previous studies using a hypomorphic model targeting huntingtin during the neurodevelopmental period indicated that loss-of-function mechanisms account for this pathogenic developmental component (Arteaga-Bracho et al., 2016). In the present study, we specifically ascertained the roles of subpallial lineage species in eliciting the previously observed HD-like phenotypes. Accordingly, we used the Cre-loxP system to conditionally ablate the murine huntingtin gene (Httflx) in cells expressing the subpallial patterning markers Gsx2 (Gsx2-Cre) or Nkx2.1 (Nkx2.1-Cre) in Httflx mice of both sexes. These genetic manipulations elicited anxiety-like behaviors, hyperkinetic locomotion, age-dependent motor deficits, and weight loss in both Httflx;Gsx2-Cre and Httflx;Nkx2.1-Cre mice. In addition, these strains displayed unique but complementary spatial patterns of basal ganglia degeneration that are strikingly reminiscent of those seen in human cases of HD. Furthermore, we observed early deficits of somatostatin-positive and Reelin-positive interneurons in both Htt subpallial null strains, as well as early increases of cholinergic interneurons, Foxp2+ arkypallidal neurons, and incipient deficits with age-dependent loss of parvalbumin-positive neurons in Httflx;Nkx2.1-Cre mice. Overall, our findings indicate that selective loss-of-huntingtin function in subpallial lineages differentially disrupts the number, complement, and survival of forebrain interneurons and globus pallidus GABAergic neurons, thereby leading to the development of key neurological hallmarks of HD during adult life. Our findings have important implications for the establishment and deployment of neural circuitries and the integrity of network reserve in health and disease.SIGNIFICANCE STATEMENT Huntington's disease (HD) is a progressive degenerative disorder caused by aberrant trinucleotide expansion in the huntingtin gene. Mechanistically, this mutation involves both loss- and gain-of-function mechanisms affecting a broad array of cellular and molecular processes. Although huntingtin is widely expressed during adult life, the mutant protein only causes the demise of selective neuronal subtypes. The mechanisms accounting for this differential vulnerability remain elusive. In this study, we have demonstrated that loss-of-huntingtin function in subpallial lineages not only differentially disrupts distinct interneuron species early in life, but also leads to a pattern of neurological deficits that are reminiscent of HD. This work suggests that early disruption of selective neuronal subtypes may account for the profiles of enhanced regional cellular vulnerability to death in HD.

Liposomal Cytarabine Induces Less Neurocognitive Dysfunction Than Intrathecal Methotrexate in an Animal Model.

Liposomal cytarabine is currently being tested clinically as an alternative to intrathecal (IT) methotrexate (MTX) for preventing relapse within the central nervous system among patients with acute lymphoblastic leukemia. To compare the toxicity and cognitive deficits caused by IT MTX versus liposomal cytarabine, juvenile Long Evans rats were treated with IT injections of MTX 1 mg/kg×4 doses over 8 days, or liposomal cytarabine 0.8 mg once. Mean concentrations of free cytarabine in cerebrospinal fluid remained above the cytotoxic threshold of 0.4 µM for 2 weeks after dosing. Animals treated with liposomal cytarabine exhibited normal recognition and spatial memory 4 weeks after injection. In contrast, exposure to IT MTX led to impaired cognitive function. In addition, mean hematocrit on day 11 was significantly lower in the MTX-treated animals (30.8%; 95% confidence interval, 27.0%-34.7%; n=6) compared with that in the liposomal cytarabine-treated animals (39.5%; 95% confidence interval, 38.4%-40.6%; n=6; P<0.0001). Our data suggest that liposomal cytarabine induces fewer neurocognitive deficits and less acute hematologic toxicity compared with IT MTX. Liposomal cytarabine may therefore have therapeutic advantages over IT MTX, if it is equally effective in preventing relapse.

Loss of Gas6 and Axl signaling results in extensive axonal damage, motor deficits, prolonged neuroinflammation, and less remyelination following cuprizone exposure.

The TAM (Tyro3, Axl, and MerTK) family of receptor tyrosine kinases (RTKs) and their ligands, Gas6 and ProS1, are important for innate immune responses and central nervous system (CNS) homeostasis. While only Gas6 directly activates Axl, ProS1 activation of Tyro3/MerTK can indirectly activate Axl through receptor heterodimerization. Therefore, we generated Gas6-/- Axl-/- double knockout (DKO) mice to specifically examine the contribution of this signaling axis while retaining ProS1 signaling through Tyro3 and MerTK. We found that naïve young adult DKO and WT mice have comparable myelination and equal numbers of axons and oligodendrocytes in the corpus callosum. Using the cuprizone model of demyelination/remyelination, transmission electron microscopy revealed extensive axonal swellings containing autophagolysosomes and multivesicular bodies, and fewer myelinated axons in brains of DKO mice at 3-weeks recovery from a 6-week cuprizone diet. Analysis of immunofluorescent staining demonstrated more SMI32+ and APP+ axons and less myelin in the DKO mice. There were no significant differences in the number of GFAP+ astrocytes or Iba1+ microglia/macrophages between the groups of mice. However, at 6-weeks cuprizone and recovery, DKO mice had increased proinflammatory cytokine and altered suppressor of cytokine signaling (SOCS) mRNA expression supporting a role for Gas6-Axl signaling in proinflammatory cytokine suppression. Significant motor deficits in DKO mice relative to WT mice on cuprizone were also observed. These data suggest that Gas6-Axl signaling plays an important role in maintaining axonal integrity and regulating and reducing CNS inflammation that cannot be compensated for by ProS1/Tyro3/MerTK signaling.

Microglial Homeostasis Requires Balanced CSF-1/CSF-2 Receptor Signaling.

CSF-1R haploinsufficiency causes adult-onset leukoencephalopathy with axonal spheroids and pigmented glia (ALSP). Previous studies in the Csf1r+/- mouse model of ALSP hypothesized a central role of elevated cerebral Csf2 expression. Here, we show that monoallelic deletion of Csf2 rescues most behavioral deficits and histopathological changes in Csf1r+/- mice by preventing microgliosis and eliminating most microglial transcriptomic alterations, including those indicative of oxidative stress and demyelination. We also show elevation of Csf2 transcripts and of several CSF-2 downstream targets in the brains of ALSP patients, demonstrating that the mechanisms identified in the mouse model are functional in humans. Our data provide insights into the mechanisms underlying ALSP. Because increased CSF2 levels and decreased microglial Csf1r expression have also been reported in Alzheimer's disease and multiple sclerosis, we suggest that the unbalanced CSF-1R/CSF-2 signaling we describe in the present study may contribute to the pathogenesis of other neurodegenerative conditions.

Methotrexate causes persistent deficits in memory and executive function in a juvenile animal model.

Methotrexate is a dihydrofolate reductase inhibitor widely employed in curative treatment for children with acute lymphoblastic leukemia (ALL). However, methotrexate administration is also associated with persistent cognitive deficits among long-term childhood cancer survivors. Animal models of methotrexate-induced cognitive deficits have primarily utilized adult animals. The purpose of present study is to investigate the neurotoxicity of methotrexate in juvenile rats and its relevant mechanisms. The doses and schedule of systemic and intrathecal methotrexate, given from post-natal age 3-7 weeks, were chosen to model the effects of repeated methotrexate dosing on the developing brains of young children with ALL. This methotrexate regimen had no visible acute toxicity and no effect on growth. At 15 weeks of age (8 weeks after the last methotrexate dose) both spatial pattern memory and visual recognition memory were impaired. In addition, methotrexate-treated animals demonstrated impaired performance in the set-shifting assay, indicating decreased cognitive flexibility. Histopathological analysis demonstrated decreased cell proliferation in methotrexate-treated animals compared to controls, as well as changes in length and thickness of the corpus callosum. Moreover, methotrexate suppressed microglia activation and RANTES production. In conclusion, our study demonstrated that a clinically relevant regimen of systemic and intrathecal methotrexate induces persistent deficits in spatial pattern memory, visual recognition memory and executive function, lasting at least 8 weeks after the last injection. The mechanisms behind methotrexate-induced deficits are likely multifactorial and may relate to suppression of neurogenesis, alterations in neuroinflammation and microglial activation, and structural changes in the corpus callosum.

Hippocampal-dependent neurocognitive impairment following cranial irradiation observed in pre-clinical models: current knowledge and possible future directions.

We reviewed the literature for studies pertaining to impaired adult neurogenesis leading to neurocognitive impairment following cranial irradiation in rodent models. This compendium was compared with respect to radiation dose, converted to equivalent dose in 2 Gy fractions (EQD2) to allow for direct comparison between studies. The effects of differences between animal species and the dependence on animal age as well as for time after irradiation were also considered. One of the major sites of de novo adult neurogenesis is the hippocampus, and as such, this review also focuses on assessing evidence related to the expression and potential effects of inflammatory cytokines on neural stem cells in the subgranular zone of the dentate gyrus and whether this correlates with neurocognitive impairment. This review also discusses potential strategies to mitigate the detrimental effects on neurogenesis and neurocognition resulting from cranial irradiation, and how the rationale for these strategies compares with the current outcome of pre-clinical studies.

A mouse model replicating hippocampal sparing cranial irradiation in humans: A tool for identifying new strategies to limit neurocognitive decline.

Cancer patients undergoing cranial irradiation are at risk of developing neurocognitive impairments. Recent evidence suggests that radiation-induced injury to the hippocampi could play an important role in this cognitive decline. As a tool for studying the mechanisms of hippocampal-dependent cognitive decline, we developed a mouse model replicating the results of the recent clinical RTOG 0933 study of hippocampal sparing whole-brain irradiation. We irradiated 16-week-old female C57BL/6J mice to a single dose of 10 Gy using either whole-brain irradiation (WBRT) or hippocampal sparing irradiation (HSI). These animals, as well as sham-irradiated controls, were subjected to behavioral/cognitive assessments distinguishing between hippocampal-dependent and hippocampal-independent functions. Irradiation was well tolerated by all animals and only limited cell death of proliferating cells was found within the generative zones. Animals exposed to WBRT showed significant deficits compared to sham-irradiated controls in the hippocampal-dependent behavioral task. In contrast, HSI mice did not perform significantly different from sham-irradiated mice (control group) and performed significantly better when compared to WBRT mice. This is consistent with the results from the RTOG 0933 clinical trial, and as such this animal model could prove a helpful tool for exploring new strategies for mitigating cognitive decline in cancer patients receiving cranial irradiation.

Memantine protects rats treated with intrathecal methotrexate from developing spatial memory deficits.

PURPOSE: To test whether memantine can prevent methotrexate-induced cognitive deficits in a preclinical model. EXPERIMENTAL DESIGN: After noting that methotrexate exposure induces prolonged elevations of the glutamate analog homocysteic acid (HCA) within cerebrospinal fluid, we tested whether intrathecal injection of HCA would produce memory deficits similar to those observed after intrathecal methotrexate. We then tested whether memantine, an antagonist of the N-methyl-d-aspartate (NMDA) subclass of glutamate receptors, could protect animals treated with clinically relevant doses of intrathecal methotrexate against developing memory deficits. Finally, we asked whether memantine affected this pathway beyond inhibiting the NMDA receptor by altering expression of the NMDA receptor or affecting concentrations of HCA or glutamate within the central nervous system. RESULTS: Four intrathecal doses of methotrexate induced deficits in spatial memory, persisting at least one month following the final injection. Intrathecal HCA was sufficient to reproduce this deficit. Concurrent administration of memantine during the period of methotrexate exposure was protective, decreasing the incidence of methotrexate-induced spatial memory deficits from 56% to 20% (P < 0.05). Memantine neither altered expression of NMDA receptors within the hippocampus nor blunted the methotrexate-induced increases in glutamate or HCA. CONCLUSIONS: Excitotoxic glutamate analogs including HCA contribute to cognitive deficits observed after intrathecal methotrexate. Memantine, an NMDA receptor antagonist, reduces the incidence of cognitive deficits in rats treated with intrathecal methotrexate, and may therefore benefit patients with cancer receiving similar treatment.

Systemic methotrexate induces spatial memory deficits and depletes cerebrospinal fluid folate in rats.

PURPOSE: Although most children with acute lymphoblastic leukemia (ALL) are cured, a subset manifests persistent, focal cognitive deficits. Methotrexate (MTX), a key component of leukemia treatment, is suspected to contribute to treatment-induced cognitive dysfunction. We sought to establish a rodent model in order to further investigate the underlying pathophysiology. PROCEDURES: Intraperitoneal MTX was given to Long-Evans rats on two schedules: acute (250 mg/kg once during adulthood), or chronic (1mg/kg twice weekly x4 doses, beginning at postnatal day 15, then weekly x6). Control rats were given saline injections on the same schedules. All male rats subsequently underwent behavioral testing designed to assess cognitive domains frequently impaired among children treated for ALL. Cerebrospinal fluid and serum folate concentrations were measured by HPLC. FINDINGS: Both acute and chronic MTX administration produced spatial memory deficits, without significantly altering visual memory, general exploration, activity or motor coordination. MTX administration was also associated with a marked reduction in serum and CSF folate and a decrease in the ratio of CSF S-adenosylmethionine to S-adenosylhomocysteine. CONCLUSIONS: Similar to children treated for ALL, rats given systemic MTX develop focal cognitive deficits along with expected alterations in folate physiology.

The chemotherapy agent, thioTEPA, yields long-term impairment of hippocampal cell proliferation and memory deficits but not depression-related behaviors in mice.

ThioTEPA is a chemotherapeutic agent used in the treatment of cancers, and more recently has been proposed as a component of high-dose therapy for young patients with recurrent malignant brain tumors. We previously demonstrated a significant dose-dependent reduction of cell proliferation in the dentate gyrus of the hippocampus in mice immediately following a 3-day regiment of thioTEPA. The aim of this study was to evaluate the long-term effects of thioTEPA treatment on hippocampal cell proliferation and potential effects on memory deficit or depression-related behavior in C57BL/6J mice. A 3-day regimen of thioTEPA (10mg/kg/d, i.p.) yielded a significant reduction in cell proliferation immediately after treatment as assessed by BrdU incorporation, and none of the labeled progeny that initially survived the treatment were detectable one week later. Following a 3-week rebound in proliferation following treatment, a significant deficit in proliferation reappeared and persisted for at least 21 weeks following treatment. ThioTEPA-treated mice subjected to an object recognition test 1, 2, 3, 4, 8, 12, 20 or 30 weeks following treatment demonstrated significant memory deficits at 12 and 20 weeks. Mice demonstrated a similar deficit in an object placement test when tested 20 weeks following thioTEPA treatment. However, no observable effects on performance in the Porsolt forced swim test or the tail suspension test were observed in thioTEPA-treated mice. Together, these studies suggest that cumulative long-term negative effects of thioTEPA treatment on proliferation of new cells in the dentate gyrus may contribute to cognitive impairments associated with its use in the treatment of cancer.