SESSION 4A TDP-43

C25 PREFERENTIAL SEQUESTRATION OF TDP-43 AND LOW MOLECULAR WEIGHT NEUROFILAMENT (NFL) MRNA TO STRESS AND DEGRADATIVE GRANULES IN AMYOTROPHIC LATERAL SCLEROSIS (ALS)

VOLKENING K, YANG W, LEYSTRA-LANTZ C, STRONG W, KAO J, STRONG M

Robarts Research Institute, London, Ontario, Canada

E-mail address for correspondence: [email protected]

Keywords: TDP-43, neurofilament, mRNA stability

Background: It has been previously shown that the steady state level of the low molecular weight neurofilament (NFL) mRNA is selectively suppressed relative to that of the remaining middle (NFM) and high (NFH) neurofilament mRNA levels in degenerating motor neurons in ALS. Because altering the stoichiometry of NFL:NFM:NFH expression is associated with neurofilament (NF) aggregate formation in transgenic mice, this has been hypothesized to contribute to the formation of pathological NF aggregates in ALS. We have shown that NFL mRNA is destabilized in ALS, and that this is due to an alteration in mRNA binding protein expression. We have previously demonstrated that the TAR DNA binding protein (TDP-43) is a NFL mRNA stability determinant and that, in contrast to healthy motor neurons in which TDP-43 is almost exclusively nuclear in its localization, wheras in ALS affected motor neurons TDP-43 is strikingly over-expressed and almost exclusively cytosolic.

Objectives: Knowing that TDP-43 is a prominent component of RNA granules, including transport, stress and degradative granules, we have examined the relative distribution of NFL mRNA and TDP-43 amongst them.

Methods: We have examined both neurologically intact (control, n = 3) and ALS lumbar spinal motor neurons (sporadic ALS, n = 3) using confocal microscopy with TDP-43 (mouse monoclonal antibody), Staufen (specific to RNA transport granules), TIA-1 (specific to stress granules) and XRN-1 (specific to RNA degradative granules). Hoescht staining was used as a nuclear staining and motor neurons identified on the basis of morphological characteristics. To determine if NFL mRNA was associated with specific RNA granules, we used immunoprecipitated (IP) staufen, TIA-1 and XRN-1, followed by RT-PCR (IP-RT-PCR) for NFL.

Results: In normal motor neurons, TDP-43 colocalized strongly with Staufen and only weakly with TIA-1 and XRN-1. In sharp contrast, in ALS affected motor neurons there was strong colocalization of TDP-43 with XRN-1, and to a lesser extent with TIA-1, suggesting that TDP-43 is being sequestered from transport granules into translationally quiescent granules. NFL mRNA was detected in all IPs except with XRN-1 in normal motor neurons. There was a statistical increase in NFL mRNA in IP-RT-PCR with TIA-1 and XRN-1 in ALS lysates.

Conclusion: These data strongly suggest that TDP-43 relocalization in ALS affects the trafficking of NFL mRNA into translationally quiescent granules, and may thus contribute to the changes in NF stoichiometry seen in ALS affected neurons. Research supported by the M. Halls Endowment.

C26 DISTRIBUTION OF TDP-43 AND UBIQUITINATED INTRACYTOPLASMIC INCLUSIONS IN THE NEUROPATHOLOGIC GRADIENT OF NEURODEGENERATION IN SALS

RAVITS J, LAURIE P, KIM Y

Benaroya Research Instituted, Seattle, WA, United States

E-mail address for correspondence: [email protected]

Keywords: TDP-43, motor neuron pathology, immunohistochemistry

Background: Motor neuron degeneration in SALS is a focal process that propagates through the neuraxis and leaves a gradient of neuron loss related to site of onset. This neuropatholgic gradient at least theoretically should represent the time-course of neurogeneration—the regions in close proximity to the site of onset in advanced stages and the more remote regions in relatively earlier stages. The distribution in this neuropathological gradient of abnormalities detected by immuno-histochemistry (IHC) with TDP-43 and ubiquitin and the correlation between the 2 antibodies are uncertain.

Objectives: i) To examine the distribution of abnormalities detected by TDP-43 and ubiquitin IHC relative to site of onset and degree of neuron loss; ii) to compare TDP-43 and ubiquitin IHC.

Methods: We had 5 groups—bulbar, arm, trunk, and leg onset disease and control. We looked at 4 neuraxis levels in each—medulla (hypoglossal nucleus), cervical cord, thoracic cord, and lumbar cord. We had 3 nervous systems in each group. We used TDP-43 and ubiquitin IHC on contiguous FFPE tissue sections. We evaluated the distribution of IHC abnormalities including the 2 morphologies (skeins and round inclusions) relative to the degree of neuron loss and compared between antibodies (TDP-43 and ubiquitin) on contiguous sections.

Results: IHC abnormalities were seen in all SALS nervous systems. IHC abnormalities was greatest in the regions remote from the onset, those with the most neurons, but this is explained by the greater number of neurons in these regions, not the frequency with which abnormalities appeared in each neuron. The abundance of IHC abnormalities between the different nervous systems within each group varied from scarce to moderate and was a variable different and independent of neuron counts. Skeins and dense round inclusions do not have obvious relation to each other: for example, skein-formation does not obviously precede round inclusions. Abnormalities detected by TDP-43 and ubiquitin have high concordance.

Discussion and Conclusions: IHC abnormalities do not clearly reflect the stage or degree of degeneration. Skeins and dense round inclusions do not clearly reflect states of protein deposition at different stages of degeneration. Allowing for extra “noise” of ubiquitin IHC, it appears that TDP-43 is the prime ubiquitinated protein depositing in motor neuron cytoplasm.The overall findings may imply that the actual death process of a particular motor neuron may be rapid (without a long process of demise) and proceed sequentially in queues rather than contemporaneously at each level as the overall degenerative process advances and summates.These findings are being quantified and are subject to change.

C27 DISTINCT PATTERNS OF TDP-43 AND PROGRANULIN EXPRESSION FOLLOWING NEURONAL INJURY

MOISSE K¹, WELCH I², VOLKENING K¹, HILL T², STRONG M¹

¹Robarts Research Institute, London, Ontario, ²The University of Western Ontario, London, Ontario, Canada

E-mail address for correspondence: [email protected]

Keywords: progranulin, TDP-43, RNA stability

Background: The nuclear factor TAR DNA-binding protein (TDP-43) has been identified as a component of ubiquitinated inclusions in degenerating spinal motor neurons in ALS. TDP-43 is unique in having dual DNA and RNA binding properties, has both nuclear export and import sequences, and has been described as a component of RNA granules. We have shown that TDP-43 regulates the stability of NFL mRNA and recently observed its inclusion in stress granules. This suggests that TDP-43 may actively participate in regulating the expression of key cytoskeletal proteins in response to neuronal injury. Understanding the normal function of TDP-43 and the effect of neuronal injury on its expression can therefore be expected to provide considerable information regarding its role in the pathogenesis of ALS.

Objectives: To determine the pattern of TDP-43 and progranulin (PGRN) expression in an acute model of neuronal injury and to compare this to the expression patterns that are widely reported for the chronic motor neuron degeneration associated with ALS.

Methods: We have performed proximal and distal sciatic axotomies in C57BL6 mice aged 6 weeks. We conducted neurobehavioural assessment and removed tissue on post-injury days 1, 3, 7, 14 and 28. We observed protein localization by immunohistochemistry, protein expression levels and cleavage states by Western blot, and mRNA expression levels by RT-PCR.

Results: Consistent with observations of TDP-43 immunoreactivity in healthy human motor neurons, TDP-43 was predominantly nuclear in motor neurons in ventral horns contralateral to the sciatic injury. Within 24 hours after injury, TDP-43 expression in ventral horns ipsilateral to axotomy was upregulated in both nuclear and cytosolic compartments, becoming maximal by day 7 and declining to contralateral levels by day 28. This pattern of increased TDP-43 protein expression, as well as increased mRNA expression, was demonstrated by Western blot and RT-PCR of lumbar spinal hemicords ipsilateral to axotomy. Of note, the converse response was observed for PGRN in which neuronal PGRN levels dramatically declined, returning to contralateral levels by day 28. Equally intriguing, the decline in neuronal PGRN expression was associated with a marked increase in microglial PGRN expression. Cytosolic TDP-43 colocalized with both staufen and TIA-1, markers for RNA transport and stress granules respectively. We did not observe colocalization of TDP-43 or PGRN with active caspase 3.

Discussion: These findings suggest that cytosolic TDP-43 may not itself be pathological, but rather a critical component of the response to neuronal injury. This is further supported by the observation that increased cytosolic TDP-43 expression and decreased neuronal PGRN expression are reversible phenomena in this acute model of neurodegeneration, suggesting that these changes are physiological responses to neuronal stress. We hypothesize that the role of TDP-43 in the cytosol is to bind, stabilize and aid in the transport of mRNA species important to the recovery of motor neurons from axotomy. Hence, the upregulation of TDP-43 expression with prominent cytosolic localization in ALS motor neurons, previously thought to be pathological, may in fact be an appropriate response to neuronal injury.

C28 TDP-43 PATHOLOGY IN ALS-LINKED MUTANT VAPB TRANSGENIC MICE

TUDOR E¹, GALTREY C ¹, LAU KF², DE VOS K¹, ACKERLEY S¹, HORTOBAGYI T, SHAW C¹, LEIGH PN¹, MILLER C¹

¹MRC Centre for Neurodegeneration Research, Institute of Psychiatry, Kings College London, United Kingdom, ²The Chinese University of Hong Kong, China

E-mail address for correspondence: [email protected]

Keywords: VAPB, TDP-43, transgenic

Background: The Pro56Ser (P56S) mutation in vesicle-associated membrane protein-associated protein B (VAPB) is a cause of some familial forms of amyotrophic lateral sclerosis (ALS). The ALS causing mutation is within the major sperm protein domain of VAPB but the mechanisms by which VABP56S induces disease are not properly understood. In order to gain insight into these mechanisms, we generated transgenic mice that overexpress wild-type VAPB (VAPBwt) or VAPBP56S.

Objectives:To investigate the mechanisms by which ALS mutant VAPBP56S induces motor neuron disease.

Methods:Transgenic mice were made that overexpress either myc-tagged VAPBwt or VAPBP56S under the control of the prion promoter, and were studied using immunohistological and biochemical methods.

Results:We generated five transgenic lines expressing VAPBwt and five lines expressing VAPBP56S. Two lines of each were selected for further study. Immunoblots revealed that both VAPBwt and VAPBP56S were expressed in spinal cord. Immunostaining with antibodies to VAPB and the myc-tag on transgene-derived VAPB revealed that both VAPBwt and VAPBP56S were present within the cytosol of motor neurons but that VAPBP56S formed aggregates. Immunostaining with antibodies to a range of ALS-associated antigens revealed the presence of ubiquitinated inclusions within the cytosol of spinal cord neurons in VAPBP56S but not VAPBwt mice. Confocal microscopy revealed that the ubiquitinated deposits co-localised with TAR DNA-binding protein 43 (TDP-43) but not VAPB. We also analysed ubiquitinated deposits in SOD1G93A transgenic mice which have been reported to not contain TDP-43 and confirmed this finding.

Discussion and Conclusions: We report ubiquitinated TDP-43 pathology in VAPBP56S transgenic mice. This is the first report of TDP-43 pathology in a transgenic model of neurodegenerative disease including ALS.

C29 UPDATE ON THE ROLE OF TDP-43 MUTATIONS AS A CAUSE OF ALS

SREEDHARAN J, ROGEL JB, MILLER CC, SHAW CE

MRC Centre for Neurodegeneration Research, Institute of Psychiatry, King's College London, United Kingdom

E-mail address for correspondence: [email protected]

Keywords: TDP-43, mutation

Background: TDP-43 was recently identified as the major protein of ubiquitinated inclusions in ∼95% of ALS cases. Following this breakthrough significant progress has been made in delineating the spectrum of “TDP-43 proteinopathies”. However, post mortem observations do not prove a biological role for TDP-43 in neurodegeneration. Our recent identification of TDP-43 mutations in sporadic (SALS) and familial (FALS) cases strongly suggests this role [1].

Objectives: To determine the mechanisms by mutant TDP-43 might contribute to neurodegeneration.

Methods: Critical analysis of recent scientific communications encompassing TDP-43 genetic and functional biological investigations.

Results: In all, 14 mutations have so far been identified in SALS and FALS cases by various groups [1–5]. These mostly cluster at the c-terminus, with one in RNA recognition motif 1 (RRM1). Several of these mutations lead to serine and threonine residue substitutions. Pathological studies of some of these cases have demonstrated TDP-43 immunoreactivity similar to that seen in SALS. This is in contrast to SOD1 FALS cases, who demonstrate a lack of TDP-43 immunoreactivity.

TDP-43 possesses both nuclear localizing (NLS) and export sequences (NES). Experimental mutation of the nuclear localizing NLS in a cell culture model resulted in mislocalisation and fragmentation of TDP-43 mimicking that seen in pathology although no toxic effect was seen [6]. However, an NLS variant was found in a FALS case with frontotemporal dementia (ALS-FTD) and shown in vitro to result in aberrant subcellular localization [7]. Toxicity of wild-type TDP-43 was demonstrated in yeast cells in which cytoplasmic aggregation of full length and select truncated versions of TDP-43 resulted in cell death [8]. Additionally, RNAi mediated TDP-43 knockdown in mammalian cells was also demonstrated to result in loss of cell membrane integrity and apoptosis [9].

Discussion and Conclusions: The clustering of TDP-43 mutations clearly implicates the c-terminus of this RNA binding protein in ALS pathogenesis. These mutations may increase the likelihood of phosphorylation or protein cleavage, but do not appear to affect nuclear localisation. The c-terminus is known to interact with other proteins, notably hnRNPs, but the complete list of interacting partners and the precise nature of these interactions is yet to be elucidated. The RRM1 mutant may be predicted to alter RNA binding, but again, the full list of RNA-interactions of TDP-43 is far from complete. The NLS mutant is perhaps the most obvious pathological candidate although it has been found in three controls to date. The possible effects of all these mutations on TDP-43 functioning and ALS pathogenesis will be elaborated on in the light of emerging evidence.