Samuel D. Crish

Distal axonopathy with structural persistence in glaucomatous neurodegeneration

An early hallmark of neuronal degeneration is distal transport loss and axon pathology. Glaucoma involves the degeneration of retinal ganglion cell (RGC) neurons and their axons in the optic nerve. Here we show that, like other neurodegenerations, distal axon injury appears early in mouse glaucoma. Where RGC axons terminate in the superior colliculus, reduction of active transport follows a retinotopic pattern resembling glaucomatous vision loss. Like glaucoma, susceptibility to transport deficits increases with age and is not necessarily associated with elevated ocular pressure. Transport deficits progress distal-to-proximal, appearing in the colliculus first followed by more proximal secondary targets and then the optic tract. Transport persists through the optic nerve head before finally failing in the retina. Although axon degeneration also progresses distal-to-proximal, myelinated RGC axons and their presynaptic terminals persist in the colliculus well after transport fails. Thus, distal transport loss is predegenerative and may represent a therapeutic target.

The microbead occlusion model: a paradigm for induced ocular hypertension in rats and mice.

PURPOSE Elevated intraocular pressure (IOP) is an important risk factor for glaucoma. Animal models often involve techniques for IOP elevation that are surgically invasive. Here the authors describe a novel and relatively simple method for inducing a highly consistent, modest, and repeatable elevation in IOP for rats and mice. METHODS IOP was elevated unilaterally by injection of polystyrene microbeads into the anterior chamber to occlude aqueous outflow in rats (2.5-7 microL) and mice (1 microL). The fellow eye received an equivalent saline injection as internal control. The authors used tonometry to measure microbead-induced IOP elevations. Optic nerves were processed histologically to determine axon loss. RESULTS For rats, a single injection of microbeads raised IOP by 21% to 34%, depending on volume, for approximately 2 weeks, though they were not tracked to full recovery. IOP in the saline-injected eye was constant. An additional injection (5 microL) extended the elevation to 8 weeks. Cumulative pressure exposure for both injections increased linearly. For mice, a single 1-microL injection of microbeads elicited a highly regular 30% elevation in IOP that persisted for more than 3 weeks, with a linear rise in cumulative pressure exposure. For both rats and mice, interanimal variability on a given day was modest, approximately 5% of the mean IOP measurement. Extended elevations (4-5 weeks) induced approximately a 20% loss of axons in both rats and mice. CONCLUSIONS These data support a novel and flexible model of modest ocular hypertension with axon loss. The maximal duration of IOP elevation will be further characterized in future studies.

Neurodegeneration in glaucoma: progression and calcium-dependent intracellular mechanisms.

Glaucoma is an age-related optic neuropathy involving sensitivity to ocular pressure. The disease is now seen increasingly as one of the central nervous system, as powerful new approaches highlight an increasing number of similarities with other age-related neurodegenerations such as Alzheimers and Parkinsons. While the etiologies of these diseases are diverse, they involve many important common elements including compartmentalized programs of degeneration targeting axons, dendrites and finally cell bodies. Most age-related degenerations display early functional deficits that precede actual loss of neuronal substrate. These are linked to several specific neurochemical cascades that can be linked back to dysregulation of Ca(2+)-dependent processes. We are now in the midst of identifying similar cascades in glaucoma. Here we review recent evidence on the pathological progression of neurodegeneration in glaucoma and some of the Ca(2+)-dependent mechanisms that could underlie these changes. These mechanisms present clear implications for efforts to develop interventions targeting neuronal loss directly and make glaucoma an attractive model for both interrogating and informing other neurodegenerative diseases.

Early Reduction of Microglia Activation by Irradiation in a Model of Chronic Glaucoma

Glaucoma is a neurodegenerative disease that results in the progressive decline and ultimate death of retinal ganglion cells (RGCs). While multiple risk factors are associated with glaucoma, the mechanisms leading to onset and progression of the disease remain unknown. Molecular analysis in various glaucoma models has revealed involvement of non-neuronal cell populations, including astrocytes, Mueller glia and microglia, at early stages of glaucoma. High-dose irradiation was reported to have a significant long-term protective effect in the DBA/2J (D2) mouse model of glaucoma, although the cellular and molecular basis for this effect remains unclear. In particular, the acute effects of irradiation on specific cell populations, including non-neuronal cells, in the D2 retina and nerve have not been assessed. Here we report that irradiation induces transient reduction in proliferating microglia within the optic nerve head and glial lamina within the first week post-irradiation. This was accompanied by reduced microglial activation, with no effect on astrocyte gliosis in those regions. At later stages we confirm that early high-dose irradiation of the mouse head results in improvement of axonal structural integrity and anterograde transport function, without reduction of intraocular pressure. Thus reduced microglial activation induced by irradiation at early stages is associated with reduced optic nerve and retinal neurodegeneration in the D2 mouse model of glaucoma.

Brimonidine prevents axonal and somatic degeneration of retinal ganglion cell neurons

BackgroundBrimonidine is a common drug for lowering ocular pressure and may directly protect retinal ganglion cells in glaucoma. The disease involves early loss of retinal ganglion cell transport to brain targets followed by axonal and somatic degeneration. We examined whether brimonidine preserves ganglion cell axonal transport and abates degeneration in rats with elevated ocular pressure induced by laser cauterization of the episcleral veins.ResultsOcular pressure was elevated unilaterally by 90% for a period of 8 weeks post- cauterization. During this time, brimonidine (1mg/kg/day) or vehicle (phosphate-buffered saline) was delivered systemically and continuously via subcutaneous pump. Animals received bilateral intravitreal injections of fluorescent cholera toxin subunit β (CTB) two days before sacrifice to assess anterograde transport. In retinas from the vehicle group, elevated pressure induced a 44% decrease in the fraction of ganglion cells with intact uptake of CTB and a 14-42% reduction in the number of immuno-labelled ganglion cell bodies, with the worst loss occurring nasally. Elevated pressure also caused a 33% loss of ganglion cell axons in vehicle optic nerves and a 70% decrease in CTB transport to the superior colliculus. Each of these components of ganglion cell degeneration was either prevented or significantly reduced in the brimonidine treatment group.ConclusionsContinuous and systemic treatment with brimonidine by subcutaneous injection significantly improved retinal ganglion cell survival with exposure to elevated ocular pressure. This effect was most striking in the nasal region of the retina. Brimonidine treatment also preserved ganglion cell axon morphology, sampling density and total number in the optic nerve with elevated pressure. Consistent with improved outcome in the optic projection, brimonidine also significantly reduced the deficits in axonal transport to the superior colliculus associated with elevated ocular pressure. As transport deficits to and from retinal ganglion cell projection targets in the brain are relevant to the progression of glaucoma, the ability of brimonidine to preserve optic nerve axons and active transport suggests its neuroprotective effects are relevant not only at the cell body, but throughout the entire optic projection.

Failure of Axonal Transport Induces a Spatially Coincident Increase in Astrocyte BDNF Prior to Synapse Loss in a Central Target

Failure of anterograde transport to distal targets in the brain is a common feature of neurodegenerative diseases. We have demonstrated in rodent models of glaucoma, the most common optic neuropathy, early loss of anterograde transport along the retinal ganglion cell (RGC) projection to the superior colliculus (SC) is retinotopic and followed by a period of persistence of RGC axon terminals and synapses through unknown molecular pathways. Here we use the DBA/2J mouse model of hereditary glaucoma and an acute rat model to demonstrate that retinotopically focal transport deficits in the SC are accompanied by a spatially coincident increase in brain-derived neurotrophic factor (BDNF), especially in hypertrophic astrocytes. These neurochemical changes occur prior to loss of RGC synapses in the DBA/2J SC. In contrast to BDNF protein, levels of Bdnf mRNA decreased with transport failure, even as mRNA encoding synaptic structures remained unchanged. In situ hybridization signal for Bdnf mRNA was the strongest in SC neurons, and labeling for the immature precursor pro-BDNF was very limited. Subcellular fractionation of SC indicated that membrane-bound BDNF decreased with age in the DBA/2J, while BDNF released from vesicles remained high. These results suggest that in response to diminished axonal function, activated astrocytes in the brain may sequester mature BDNF released from target neurons to counter stressors that otherwise would challenge survival of projection synapses.

Proximal Inhibition of p38 MAPK Stress Signaling Prevents Distal Axonopathy

The p38 mitogen-activated protein kinase (MAPK) isoforms are phosphorylated by a variety of stress stimuli in neurodegenerative disease and act as upstream activators of myriad pathogenic processes. Thus, p38 MAPK inhibitors are of growing interest as possible therapeutic interventions. Axonal dysfunction is an early component of most neurodegenerative disorders, including the most prevalent optic neuropathy, glaucoma. Sensitivity to intraocular pressure at an early stage disrupts anterograde transport along retinal ganglion cell (RGC) axons to projection targets in the brain with subsequent degeneration of the axons themselves; RGC body loss is much later. Here we show that elevated ocular pressure in rats increases p38 MAPK activation in retina, especially in RGC bodies. Topical eye-drop application of a potent and selective inhibitor of the p38 MAPK catalytic domain (Ro3206145) prevented both the degradation of anterograde transport to the brain and degeneration of axons in the optic nerve. Ro3206145 reduced in the retina phosphorylation of tau and heat-shock protein 27, both down-stream targets of p38 MAPK activation implicated in glaucoma, as well as expression of two inflammatory responses. We also observed increased p38 MAPK activation in mouse models. Thus, inhibition of p38 MAPK signaling in the retina may represent a therapeutic target for preventing early pathogenesis in optic neuropathies.

Anterograde transport blockade precedes deficits in retrograde transport in the visual projection of the DBA/2J mouse model of glaucoma.

Axonal transport deficits have been reported as an early pathology in several neurodegenerative disorders, including glaucoma. However, the progression and mechanisms of these deficits are poorly understood. Previous work suggests that anterograde transport is affected earlier and to a larger degree than retrograde transport, yet this has never been examined directly in vivo. Using combined anterograde and retrograde tract tracing methods, we examined the time-course of anterograde and retrograde transport deficits in the retinofugal projection in pre-glaucomatous (3 month-old) and glaucomatous (9–13 month old) DBA/2J mice. DBA/2J-Gpnmb+ mice were used as a control strain and were shown to have similar retinal ganglion cell densities as C57BL/6J control mice—a strain commonly investigated in the field of vision research. Using cholera toxin-B injections into the eye and FluoroGold injections into the superior colliculus (SC), we were able to measure anterograde and retrograde transport in the primary visual projection. In DBA/2J, anterograde transport from the retina to SC was decreased by 69% in the 9–10 month-old age group, while retrograde transport was only reduced by 23% from levels seen in pre-glaucomatous mice. Despite this minor reduction, retrograde transport remained largely intact in these glaucomatous age groups until 13-months of age. These findings indicate that axonal transport deficits occur in semi-functional axons that are still connected to their brain targets. Structural persistence as determined by presence of estrogen-related receptor beta label in the superficial SC was maintained beyond time-points where reductions in retrograde transport occurred, also supporting that transport deficits may be due to physiological or functional abnormalities as opposed to overt structural loss.

TREM2 deficiency exacerbates tau pathology through dysregulated kinase signaling in a mouse model of tauopathy

BackgroundGenetic variants of the Triggering Receptor Expressed on Myeloid Cells-2 (TREM2) confer increased risk of developing late-onset Alzheimer’s Disease (LOAD) and other neurodegenerative disorders. Recent studies provided insight into the multifaceted roles of TREM2 in regulating extracellular β-amyloid (Aβ) pathology, myeloid cell accumulation, and inflammation observed in AD, yet little is known regarding the role of TREM2 in regulating intracellular microtubule associated protein tau (MAPT; tau) pathology in neurodegenerative diseases and in AD, in particular.ResultsHere we report that TREM2 deficiency leads to accelerated and exacerbated hyperphosphorylation and aggregation of tau in a humanized mouse model of tauopathy. TREM2 deficiency also results, indirectly, in dramatic widespread dysregulation of neuronal stress kinase pathways.ConclusionsOur results suggest that deficiency of microglial TREM2 leads to heightened tau pathology coupled with widespread increases in activated neuronal stress kinases. These findings offer new insight into the complex, multiple roles of TREM2 in regulating Aβ and tau pathologies.

Altered Neuroinflammation and Behavior after Traumatic Brain Injury in a Mouse Model of Alzheimer's Disease

Traumatic brain injury (TBI) has acute and chronic sequelae, including an increased risk for the development of Alzheimers disease (AD). TBI-associated neuroinflammation is characterized by activation of brain-resident microglia and infiltration of monocytes; however, recent studies have implicated beta-amyloid as a major manipulator of the inflammatory response. To examine neuroinflammation after TBI and development of AD-like features, these studies examined the effects of TBI in the presence and absence of beta-amyloid. The R1.40 mouse model of cerebral amyloidosis was used, with a focus on time points well before robust AD pathologies. Unexpectedly, in R1.40 mice, the acute neuroinflammatory response to TBI was strikingly muted, with reduced numbers of CNS myeloid cells acquiring a macrophage phenotype and decreased expression of inflammatory cytokines. At chronic time points, macrophage activation substantially declined in non-Tg TBI mice; however, it was relatively unchanged in R1.40 TBI mice. The persistent inflammatory response coincided with significant tissue loss between 3 and 120 days post-injury in R1.40 TBI mice, which was not observed in non-Tg TBI mice. Surprisingly, inflammatory cytokine expression was enhanced in R1.40 mice compared with non-Tg mice, regardless of injury group. Although R1.40 TBI mice demonstrated task-specific deficits in cognition, overall functional recovery was similar to non-Tg TBI mice. These findings suggest that accumulating beta-amyloid leads to an altered post-injury macrophage response at acute and chronic time points. Together, these studies emphasize the role of post-injury neuroinflammation in regulating long-term sequelae after TBI and also support recent studies implicating beta-amyloid as an immunomodulator.